Enzymatically active modified subtilisins

ABSTRACT

Novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and DNA sequences encoding the same. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in the amino acid sequence of a precursor carbonyl hydrolase. Such mutants have one or more properties which are different than the same property of the precursor hydrolase.

This is a continuation of application Ser. No. 07/898,382 filed Jun. 9, 1992, now abandoned, which is a continuation of application Ser. No. 07/747,459 filed Aug. 12, 1991 now abandoned, which is a continuation of 07/540,868, filed Jun. 14, 1990 now abandoned, which is a continuation of application Ser. No. 07/035,652 filed Apr. 6, 1987, now abandoned, which is a continuation-in-part of application Ser. No. 06/858,594 filed Apr. 30, 1986, now abandoned , which is a continuation-in-part of application Ser. No. 06/614,612 filed May 29, 1984, now abandoned, which issued as U.S. Pat. No. 4,760,025 on Jul. 28, 1988, which is a continuation-in-part of application Ser. No. 06/614,615 filed May 29, 1984, now abandoned, which is a continuation-in-part of application Ser. No. 06/614,617 filed May 29, 1984, now abandoned, which is a continuation-in-part of application Ser. No. 06/614,491 filed May 29, 1984, and now abandoned, each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and to DNA sequences encoding the same. Such mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in a precursor amino acid sequence.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolase. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. M. (1974) Sci Amer. 131, 74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: the Bacillus subtilisins and the mammalian and homologous bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.

Subtilisin is a serine endoprotease (MW 27,500) which is secreted in large amounts from a wide variety of Bacillus species. The protein sequence of subtilisin has been determined from at least four different species of Bacillus. Markland, F. S., et al. (1971) in The Enzymes, ed. Boyer P. D., Acad Press, New York, Vol. III, pp. 561-608; Nedkov, P. et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 1537-1540. The three-dimensional crystallographic structure of subtilisin BPN' (from B. amyloligoefaciens) to 2.5 Å resolution has also been reported. Wright, C. S., et al. (1969) Nature 221, 235-242; Drenth, J. et al. (1972) Eur. J. Biochem. 26, 177-181. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972) Biochemistry 11, 2439-2449), product complexes (Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), and transition state analogs (Matthews, D. A., et al (1975) J. Biol. Chem. 250, 7120-7126; Poulos, T. L., et al. (1976) J. Biol. Chem. 251, 1097-1103), which have been reported have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin (Philipp, M., et al. (1983) Mol. Cell. Biochem. 51, 5-32; Svendsen, I. B. (1976) Carlsbera Res. Comm. 41, 237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methionine at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965) J. Biol. Chem. 244, 5333-5338).

Substrate specificity is a ubiquitous feature of biological macromolecules that is determined by chemical forces including hydrogen bonding, electrostatic, hydrophobic and steric interactions. Jencks, W. P., in Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., in Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287. Substrate specificity studies of enzymes, however, have been limited to the traditional means of probing the relative importance of these binding forces. Although substrate analogs can be synthesized chemically, the production of modified enzyme analogs has been limited to chemically modified enzyme derivatives (Kaiser, E. T., et al. (1985) Ann. Rev. Biochem. 54, 565-595 or naturally occurring mutants. Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358.

The recent development of various in vitro techniques to manipulate the DNA sequences encoding naturally-occuring polypeptides as well as recent developments in the chemical synthesis of relatively short sequences of single and double stranded DNA has resulted in the speculation that such techniques can be used to modify enzymes to improve some functional property in a predictable way. Ulmer, K. M. (1983) Science 219, 666-671. The only working example disclosed therein, however, is the substitution of a single amino acid within the active site of tyrosyl-tRNA synthetase (Cys35→Ser) which lead to a reduction in enzymatic activity. See Winter, G., et al. (1982) Nature 299, 756-758; and Wilkinson, A. J., et al. (1983) Biochemistry 22, 3581-3586 (Cys35-Gly mutation also resulted in decreased activity).

When the same t-RNA synthetase was modified by substituting a different amino acid residue within the active site with two different amino acids, one of the mutants (Thr51→Ala) reportedly demonstrated a predicted moderate increase in kcat/K_(M) whereas a second mutant (Thr51→Pro) demonstrated a massive increase in kcat/K_(M) which could not be explained with certainty. Wilkinson, A. H., et al. (1984) Nature 307, 187-188.

Another reported example of a single substitution of an amino acid residue is the substitution of cysteine for isoleucine at the third residue of T4 lysozyme. Perry, L. J., et al. (1984) Science 226, 555-557. The resultant mutant lysozyme was mildly oxidized to form a disulfide bond between the new cysteine residue at position 3 and the native cysteine at position 97. This crosslinked mutant was initially described by the author as being enzymatically identical to, but more thermally stable than, the wild type enzyme. However, in a "Note Added in Proof", the author indicated that the enhanced stability observed was probably due to a chemical modification of cysteine at residue 54 since the mutant lysozyme with a free thiol at Cys54 has a thermal stability identical to the wild type lysozyme. Similarly, a modified dihydrofolate reductase from E.coli has been reported to be modified by similar methods to introduce a cysteine which could be crosslinked with a naturally-occurring cysteine in the reductase. Villafranca, D. E., et al. (1983) Science 222, 782-788. The author indicates that this mutant is fully reactive in the reduced state but has significantly diminished activity in the oxidized state. In addition, two other substitutions of specific amino acid residues are reported which resulted in mutants which had diminished or no activity.

As set forth below, several laboratories have also reported the use of site directed mutagenesis to produce the mutation of more than one amino acid residue within a polypeptide.

The amino-terminal region of the signal peptide of the prolipoprotein of the E. coli outer membrane was stated to be altered by the substitution or deletion of residues 2 and 3 to produce a charge change in that region of the polypeptide. Inoyye, S., et al. (1982) Proc. Nat. Acad. Sci. USA 79, 3438-3441. The same laboratory also reported the substitution and deletion of amino acid residues 9 and 14 to determine the effects of such substitution on the hydrophobic region of the same signal sequence. Inouye, S., et al. (1984) J. Biol. Chem. 259, 3729-3733. In the case of mutants at residues 2 and 3 the authors state that the results obtained were consistant with the proposed loop model for explaining the functions of the signal sequence. However, as reported the mutations at residues 9 and 14 produced results indicating that the signal peptide has unexpected flexibility in terms of the relationship between its primary structure and function in protein secretion.

Double mutants in the active site of tyrosyl-t-RNA synthetase have also been reported. Carter, P. J., et al. (1984) Cell 38, 835-840. In this report, the improved affinity of the previously described Thr51Pro mutant for ATP was probed by producing a second mutation in the active site of the enzyme. One of the double mutants, Gly35/Pro51, reportedly demonstrated an unexpected result in that it bound ATP in the transition state better than was expected from the two single mutants. Moreover, the author warns, at least for one double mutant, that it is not readily predictable how one substitution alters the effect caused by the other substitution and that care must be taken in interpreting such substitutions.

A mutant is disclosed in U.S. Pat. No. 4,532,207, wherein a polyarginine tail was attached to the C-terminal residue of β-urogastrone by modifying the DNA sequence encoding the polypeptide. As disclosed, the polyarginine tail changed the electrophoretic mobility of the urogastrone-polyarginine hybrid permiting selective purification. The polyarginine was subsequently removed, according to the patentee, by a polyarginine specific exopeptidase to produce the purified urogastrone. Properly construed, this reference discloses hybrid polypeptides which do not constitute mutant polypeptides containing the substitution, insertion or deletion of one or more amino acids of a naturally occurring polypeptide. Single and double mutants of rat pancreatic trypsin have also been reported. Craik, C. S., et al. (1985) Science 228, 291-297. As reported, glycine residues at positions 216 and 226 were replaced with alanine residues to produce three trypsin mutants (two single mutants and one double mutant). In the case of the single mutants, the authors stated expectation was to observe a differential effect on K_(M). They instead reported a change in specificity (kcat/K_(M)) which was primarily the result of a decrease in kcat. In contrast, the double mutant reportedly demonstrated a differential increase in K_(M) for lysyl and arginyl substrates as compared to wild type trypsin but had virtually no catalytic activity.

The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications.

Based on the above references, however, it is apparent that the modification of the amino acid sequence of wild type enzymes often results in the decrease or destruction of biological activity. Moreover, these references do not address the mutation of the particular carbonyl hydrolases disclosed herein.

Accordingly, it is an object herein to provide carbonyl hydrolase mutants which have at least one property which is different from the same property of the carbonyl hydrolase precursor from which the amino acid of said mutant is derived.

It is a further object to provide mutant DNA sequences encoding such carbonyl hydrolase mutants as well as expression vectors containing such mutant DNA sequences.

Still further, another object of the present invention is to provide host cells transformed with such vectors as well as host cells which are capable of expressing such mutants either intracellularly or extracellularly.

SUMMARY OF THE INVENTION

The invention includes carbonyl hydrolase mutants, preferably having at least one property which is substantially different from the same property of the precursor non-human carbonyl hydrolase from which the amino acid sequence of the mutant is derived. These properties include oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile and resistance to proteolytic degradation. The precursor carbonyl hydrolases may be naturally occurring carbonyl hydrolases or recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is derived by the substitution, deletion or insertion of one or more amino acids of the precursor carbonyl hydrolase amino acid sequence.

The invention also includes mutant DNA sequences encoding such carbonyl hydrolase mutants. These mutant DNA sequences are derived from a precursor DNA sequence which encodes a naturally occurring or recombinant precursor carbonyl hydrolase. The mutant DNA sequence is derived by modifying the precursor DNA sequence to encode the substitution, deletion or insertion of one or more amino acids encoded by the precursor DNA sequence. These recombinant DNA sequences encode mutants having an amino acid sequence which does not exist in nature and at least one property which is substantially different from the same property of the precursor carbonyl hydrolase encoded by the precursor DNA sequence.

Further the invention includes expression vectors containing such mutant DNA sequences as well as host cells transformed with such vectors which are capable of expressing said carbonyl hydrolase mutants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the coding strand, correlated with the amino acid sequence of B. amyloliquefaciens subtilisin gene. Promoter (p) ribosome binding site (rbs) and termination (term) regions of the DNA sequence as well as sequences encoding the presequence (PRE) putative prosequence (PRO) and mature form (MAT) of the hydrolase are also shown.

FIG. 2 is a schematic diagram showing the substrate binding cleft of subtilisin together with substrate.

FIG. 3 is a stereo view of the S-1 binding subsite of B. amyloliquefaciens subtilisin showing a lysine P-1 substrate bound in the site in two different ways. FIG. 3A shows Lysine P-1 substrate bound to form a salt bridge with a Glu at position 156. FIG. 3B shows Lysine P-1 substrate bound to form a salt bridge with Glu at position 166.

FIG. 4 is a schematic diagram of the active site of subtilisin Asp32, His64 and Ser221.

FIGS. 5A-1, 5A-2, 5B-1, 5B-2 and 5C depict the amino acid sequence of subtilisin obtained from various sources. The residues directly beneath each residue of B. amyloliquefaciens subtilisin are equivalent residues which (1) can be mutated in a similar manner to that described for B. amyloliquefaciens subtilisin, or (2) can be used as a replacement amino acid residue in B. amyloliquefaciens subtilisin. FIG. 5C depicts conserved residues of B. amyloliquefaciens subtilisin when compared to other subtilisin sequences.

FIGS. 6A and 6B depict the inactivation of the mutants Met222L and Met222Q when exposed to various organic oxidants.

FIGS. 7A-7B depicts the ultraviolet spectrum of Met222F subtilisin and the difference spectrum generated after inactivation by diperdodecanoic acid (DPDA).

FIG. 8 shows the pattern of cyanogen bromide digests of untreated and DPDA oxidized subtilisin Met222F on high resolution SDS-pyridine peptide gels.

FIG. 9 depicts a map of the cyanogen bromide fragments of FIG. 8 and their alignment with the sequence of subtilisin Met222F.

FIG. 10 depicts the construction of mutations between codons 45 and 50 of B. amyloliquefaciens subtilisin.

FIG. 11 depicts the construction of mutations between codons 122 and 127 of B. amyloliquefaciens subtilisin.

FIG. 12 depicts the effect of DPDA on the activity of subtilisin mutants at positions 50 and 124 in subtilisin Met222F.

FIG. 13 depicts the construction of mutations at codon 166 of B. amyloliquefaciens subtilisin.

FIG. 14 depicts the effect of hydrophobicity of the P-1 substrate side-chain on the kinetic parameters of wild-type B. amyloliquefaciens subtilisin.

FIGS. 15A and 15B depicts the effect of position 166 side-chain substitutions on P-1 substrate specificity. FIG. 15A shows position 166 mutant subtilisins containing non-branched alkyl and aromatic side-chain substitutions arranged in order of increasing molecular volume. FIG. 15B shows a series of mutant enzymes progressing through β- and γ-branched aliphatic side chain substitutions of increasing molecular volume.

FIGS. 16A, 16B, 16C and 16D depicts the effect of position 166 side-chain volume on log kcat/K_(M) for various P-1 substrates.

FIG. 17 shows the substrate specificity differences between Ile166 and wild-type (Gly166) B. amyloliquefaciens subtilisin against a series of aliphatic and aromatic substrates. Each bar represents the difference in log kcat/K_(M) for Ile166 minus wild-type (Gly166) subtilisin.

FIG. 18 depicts the construction of mutations at codon 169 of B. amyloliquefaciens subtilisin.

FIG. 19 depicts the construction of mutations at codon 104 of B. amyloliquefaciens subtilisin.

FIG. 20 depicts the construction of mutations at codon 152 B. amyloliquefaciens subtilisin.

FIG. 21 depicts the construction of single mutations at codon 156 and double mutations at codons 156 and 166 of B. amyloliquefaciens subtilisin.

FIG. 22 depicts the construction of mutations at codon 217 for B. amyloliquefaciens subtilisin.

FIG. 23A depicts the kcat/K_(M) versus pH profile for mutations at codon 156 and 166 in B. amyloliquefaciens subtilisin.

FIG. 23B depicts the kcat/K_(M) versus pH profile for mutations at codon 156 and 166 in B. amyloliquefaciens subtilisin.

FIG. 24 depicts the kcat/K_(M) versus pH profile for mutations at codon 222 in B. amyloliquefaciens subtilisin.

FIG. 25 depicts the construction of mutants at codons 94, 95 and 96.

FIGS. 26 and 27 depict substrate specificity of proteins for 4 substrates.

FIGS. 28 A, B, C and D depict the effect of charge in the P-1 binding sites due to substitutions at codon 156 and 166.

FIGS. 29 A and B are a stereoview of the P-1 binding site of subtilisin BPN' showing a lysine P-1 substrate bound in the site in two ways. In 29A, Lysine P-1 substrate is built to form a salt bridge with a Glu at codon 156. In 29B, Lysine P-1 substrate is built to form a salt bridge with Glu at codon 166.

FIGS. 30A, 30B, and 30C demonstrates residual enzyme activity versus temperature curves for purified wild-type (Panel A) C22/C87 (Panel B) and C24/C87 (Panel C).

FIG. 31 depicts the strategy for producing point mutations in the subtilisin coding sequence by misincorporation of α-thioldeoxynucleotide triphosphates.

FIG. 32 depicts the autolytic stability of purified wild type and mutant subtilisins 170E, 107V, 213R and 107V/213R at alkaline pH.

FIG. 33 depicts the autolytic stability of purified wild type and mutant subtilisins V50, F50 and F50/V107/R213 at alkaline pH.

FIG. 34 depicts the strategy for constructing plasmids containing random cassette mutagenesis over residues 197 through 228.

FIG. 35A depicts the oligodeoxynucleotides used for random cassette mutagenesis over residues 197 through 228.

FIG. 36 depicts the construction of mutants at codon 204.

FIG. 37 depicts the oligodeoxynucleotides used for synthesizing mutants at codon 204.

DETAILED DESCRIPTION

The inventors have discovered that various single and multiple in vitro mutations involving the substitution, deletion or insertion of one or more amino acids within a non-human carbonyl hydrolase amino acid sequence can confer advantageous properties to such mutants when compared to the non-mutated carbonyl hydrolase.

Specifically, B. amyloliquefaciens subtilisin, an alkaline bacterial protease, has been mutated by modifying the DNA encoding the subtilisin to encode the substitution of one or more amino acids at various amino acid residues within the mature form of the subtilisin molecule. These in vitro mutant subtilisins have at least one property which is different when compared to the same property of the precursor subtilisin. These modified properties fall into several categories including: oxidative stability, substrate specificity, thermal stability, alkaline stability, catalytic activity, pH activity profile, resistance to proteolytic degradation, K_(M), kcat and K_(M) /kcat ratio.

Carbonyl hydrolases are enzymes which hydrolyze

compounds containing ##STR1## in which X is oxygen or nitrogen. They include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. Naturally occurring carbonyl hydrolases principally include hydrolases, e.g. lipases and peptide hydrolases, e.g. subtilisins or metalloproteases. Peptide hydrolases include α-aminoacylpeptide hydrolase, peptidylamino-acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid proteases are included, as well as endo and exoproteases.

"Recombinant carbonyl hydrolase" refers to a carbonyl hydrolase in which the DNA sequence encoding the naturally occurring carbonyl hydrolase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the carbonyl hydrolase amino acid sequence. Suitable modification methods are disclosed herein and in EPO Publication No. 0130756 published Jan. 9, 1985.

Subtilisins are bacterial carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "subtilisin" means a naturally occurring subtilisin or a recombinant subtilisin. A series of naturally occurring subtilisins is known to be produced and often secreted by various bacterial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus is aspartate-histidineserine. In the chymotrypsin related proteases the relative order, however is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.

"Recombinant subtilisin" refers to a subtilisin in which the DNA sequence encoding the subtilisin is modified to produce a mutant DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally occurring subtilisin amino acid sequence. Suitable methods to produce such modification include those disclosed herein and in EPO Publication No. 0130756. For example, the subtilisin multiple mutant herein containing the substitution of methionine at amino acid residues 50, 124 and 222 with phenylalanine, isoleucine and glutamine, respectively, can be considered to be derived from the recombinant subtilisin containing the substitution of glutamine at residue 222 (Gln222) disclosed in EPO Publication No. 0130756. The multiple mutant thus is produced by the substitution of phenylalanine for methionine at residue 50 and isoleucine for methionine at residue 124 in the Gln222 recombinant subtilisin.

"Non-human carbonyl hydrolases" and their genes may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include Gram negative organisms such as E. coli or pseudomonas and Gram positive bacteria such as micrococcus or bacillus. Examples of eucaryotic organisms from which carbonyl hydrolase and their genes may be obtained include yeast such as S. cerevisiae, fungi such as Aspergillus sp., and non-human mammalian sources such as, for example, Bovine sp. from which the gene encoding the carbonyl hydrolase chymosin can be obtained. As with subtilisins, a series of carbonyl hydrolases can be obtained from various related species which have amino acid sequences which are not entirely homologous between the members of that series but which nevertheless exhibit the same or similar type of biological activity. Thus, non-human carbonyl hydrolase as used herein has a functional definition which refers to carbonyl hydrolases which are associated, directly or indirectly, with procaryotic and non-human eucaryotic sources.

A "carbonyl hydrolase mutant" has an amino acid sequence which is derived from the amino acid sequence of a non-human "precursor carbonyl hydrolase". The precursor carbonyl hydrolases include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is "derived" from the precursor hydrolase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the "precursor DNA sequence" which encodes the amino acid sequence of the precursor carbonyl hydrolase rather than manipulation of the precursor carbonyl hydrolase per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in EPO Publication No. 0130756.

Specific residues of B. amyloliquefaciens subtilisin are identified for substitution, insertion or deletion. These amino acid position numbers refer to those assigned to the B. amyloliquefaciens subtilisin sequence presented in FIG. 1. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor carbonyl hydrolases containing amino acid residues which are "equivalent" to the particular identified residues in B. amyloliquefaciens subtilisin.

A residue (amino acid) of a precursor carbonyl hydrolase is equivalent to a residue of B. amyloliquefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analagous to a specific residue or portion of that residue in B. amyloliquefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor carbonyl hydrolase is directly compared to the B. amyloliquefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in all subtilisins for which sequence is known (FIG. 5C). After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of B. amyloliquefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained.

For example, in FIGS. 5A-1 and 5A-2 the amino acid sequence of subtilisin from B. amyloliquefaciens, B. subtilisin var. I168 and B. lichinformis (carlsbergensis) are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. These residues are identified in FIG. 5C.

These conserved residues thus may be used to define the corresponding equivalent amino acid residues of B. amyloliquefaciens subtilisin in other carbonyl hydrolases such as thermitase derived from Thermoactinomyces. These two particular sequences are aligned in FIG. 5B to produce the maximum homology of conserved residues. As can be seen there are a number of insertions and deletions in the thermitase sequence as compared to B. amyloliquefaciens subtilisin. Thus, the equivalent amino acid of Tyr217 in B. amyloliquefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217.

In FIG. 5A, the equivalent amino acid at position 217 in B. amyloliquefaciens subtilisin is Tyr. Likewise, in B. subtilis subtilisin position 217 is also occupied by Tyr but in B. licheniformis position 217 is occupied by Leu.

Thus, these particular residues in thermitase, and subtilisin from B. subtilisin and B. licheniformis may be substituted by a different amino acid to produce a mutant carbonyl hydrolase since they are equivalent in primary structure to Tyr217 in B. amyloliquefaciens subtilisin. Equivalent amino acids of course are not limited to those for Tyr217 but extend to any residue which is equivalent to a residue in B. amyloliquefaciens whether such residues are conserved or not.

Equivalent residues homologous at the level of tertiary structure for a precursor carbonyl hydrolase whose tertiary structure has been determined by x-ray crystallography, are defined as those for which the atomic coordinates of 2 or more of the main chain atoms of a particular amino acid residue of the precursor carbonyl hydrolase and B. amyloliquefaciens subtilisin (N on N, CA on CA, C on C, and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the carbonyl hydrolase in question to the B. amyloliquefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. ##EQU1## Equivalent residues which are functionally analogous to a specific residue of B. amyloliquefaciens subtilisin are defined as those amino acids of the precursor carbonyl hydrolases which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the B. amyloliquefaciens subtilisin as described herein. Further, they are those residues of the precursor carbonyl hydrolase (for which a tertiary structure has been obtained by x-ray crystallography), which occupy an analogous position to the extent that although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of B. amyloliquefaciens subtilisin. The three dimensional structures would be aligned as outlined above.

Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a mutant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in a naturally occurring sequence. The carbonyl hydrolase mutants of the present invention include the mature forms of carbonyl hydrolase mutants as well as the pro- and prepro-forms of such hydrolase mutants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the carbonyl hydrolase mutants.

"Prosequence" refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a carbonyl hydrolase which when removed results in the appearance of the "mature" form of the carbonyl hydrolase. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. The preferred prosequence for producing carbonyl hydrolase mutants, specifically subtilisin mutants, is the putative prosequence of B. amyloliquefaciens subtilisin although other subtilisin prosequences may be used.

A "signal sequence" or "presequence" refers to any sequence of amino acids bound to the N-terminal portion of a carbonyl hydrolase or to the N-terminal portion of a prohydrolase which may participate in the secretion of the mature or pro forms of the hydrolase. This definition of signal sequence is a functional one, meant to include all those amino acid sequences, encoded by the 5'-terminal portion of the subtilisin gene or other secretable carbonyl hydrolases, which participate in the effectuation of the secretion of subtilisin or other carbonyl hydrolases under native conditions. The present invention utilizes such sequences to effect the secretion of the carbonyl hydrolase mutants as defined herein.

A "prepro" form of a carbonyl hydrolase mutant consists of the mature form of the hydrolase having a prosequence operably linked to the amino-terminus of the hydrolase and a "pre" or "signal" sequence operably linked to the amino terminus of the prosequence.

"Expression vector" refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid" and "vector" are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

The "host cells" used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in EPO Publication No. 0130756 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing subtilisin is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in EPO Publication No. 0130756 and further described by Yang, M. Y., et al. (1984) J. Bacteriol. 160, 15-21. Such host cells are distinguishible from those disclosed in PCT Publication No. 03949 wherein enzymatically inactive mutants of intracellular proteases in E. coli are disclosed. Other host cells for expressing subtilisin include Bacillus subtilis I168 (EPO Publication No. 0130756).

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the carbonyl hydrolase mutants or expressing the desired carbonyl hydrolase mutant. In the case of vectors which encode the pre or prepro form of the carbonyl hydrolase mutant, such mutants, when expressed, are typically secreted from the host cell into the host cell medium.

"Operably linked" when describing the relationship between two DNA regions simply means that they are functional ly related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. The genes encoding the naturally-occurring precursor carbonyl hydrolase may be obtained in accord with the general methods described in EPO Publication No. 0130756. As can be seen from the examples disclosed therein, the methods generally comprise synthesizing labelled probes having putative sequences encoding regions of the hydrolase of interest, preparing genomic libraries from organisms expressing the hydrolase, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The cloned carbonyl hydrolase is then used to transform a host cell in order to express the hydrolase. The hydrolase gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promotor if it is recognized, i.e., transcribed, by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the hydrolase gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the hydrolase gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosonal limitations. However, it is within the scope herein to integrate multiple copies of the hydrolase gene into host genome. This is facilitated by procaryotic and eucaryotic organisms which are particularly susceptible to homologous recombination.

Once the carbonyl hydrolase gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor carbonyl hydrolase. Such modifications include the production of recombinant carbonyl hydrolases as disclosed in EPO Publication No. 0130756 and the production of carbonyl hydrolase mutants described herein.

The following cassette mutagenesis method may be used to facilitate the construction and identification of the carbonyl hydrolase mutants of the present invention although other methods including site-directed mutagenesis may be used. First, the gene encoding the hydrolase is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the expressed enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the hydrolase gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the hydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.

Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. Once the gene is cloned, the restriction sites flanking the sequence to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is enormously simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

The number of commercially available restriction enzymes having sites not present in the gene of interest is generally large. A suitable DNA sequence computer search program simplifies the task of finding potential 5' and 3' convenient flanking sites. A primary constraint is that any mutation introduced in creation of the restriction site must be silent to the final construction amino acid coding sequence. For a candidate restriction site 5' to the target codon a sequence must exist in the gene which contains at least all the nucleotides but for one in the recognition sequence 5' to the cut of the candidate enzyme. For example, the blunt cutting enzyme SmaI (CCC/GGG) would be a 5' candidate if a nearby 5' sequence contained NCC, CNC, or CCN. Furthermore, if N needed to be altered to C this alteraiton must leave the amino acid coding sequence intact. In cases where a permanent silent mutation is necessary to introduce a restriction site one may want to avoid the introduction of a rarely used codon. A similar situation of SmaI would apply for 3' flanking sites except the sequence NGG, GNG, or GGN must exist. The criteria for locating candidate enzymes is most relaxed for blunt cutting enzymes and most stringent for 4 base overhang enzymes. In general many candidate sites are available. For the codon-221 target described herein a BalI site (TGG/CCA) would have been engineered in one base pair 5' from the KpnI site. A 3' EcoRV site (GAT/ATC) could have been employed 11 base pairs 5' to the PstI site. A cassette having termini ranging from a blunt end up to a four base-overhang will function without difficulty. In retrospect, this hypothetical EcoRV site would have significantly shortened the oligonucleotide cassette employed (9 and 13 base pairs) thus allowing greater purity and lower pool bias problems. Flanking sites should obviously be chosen which cannot themselves ligate so that ligation of the oligonucleotide cassette can be assured in a single orientation.

The mutant carbonyl hydrolases expressed upon transformation of suitable hosts are screened for enzymes exhibiting one or more properties which are substantially different from the properties of the precursor carbonyl hydrolases, e.g., changes in substrate specificity, oxidative stability, thermal stability, alkaline stability, resistance to proteolytic degradation, pH-activity profiles and the like.

The carbonyl hydrolase mutants of the present invention may also be generated by random mutagenesis. See for example the methods disclosed by Shortle, D., et al. (1985) Genetics, 110, 539; Shortle, D., et al. (1986) Proteins: Structure, Function and Genetics, 1, 81; Shortle, D. (1986) J. Cell. Biochem, 30, 281; Alber, T., et al. (1985) Proc. Natl. Acad. of Sci., 82, 747; Matsumura, M., et al. (1985) J. Biochem., 260, 15298; Liao, H., et al. (1986) Proc. Natl. Acad. of Sci., 83 576; and the random mutagenesis method disclosed herein.

When combined with the alkaline stability screening procedure disclosed herein, mutants obtained by random mutagenesis were identified which demonstrated either increased or decreased alkaline or thermal stability.

A change in substrate specificity is defined as a difference between the kcat/K_(M) ratio of the precursor carbonyl hydrolase and that of the hydrolase mutant. The kcat/K_(M) ratio is a measure of catalytic efficienty. Carbonyl hydrolase mutants with increased or diminished kcat/K_(M) ratios are described in the examples. Generally, the objective will be to secure a mutant having a greater (numerically large) kcat/K_(M) ratio for a given substrate, thereby enabling the use of the enzyme to more efficiently act on a target substrate. A substantial change in kcat/K_(M) ratio is preferably at least 2-fold increase or decrease. However, smaller increases or decreases in the ratio (e.g., at least 1.5-fold) are also considered substantial. An increase in kcat/K_(M) ratio for one substrate may be accompanied by a reduction in kcat/K_(M) ratio for another substrate. This is a shift in substrate specificity, and mutants exhibiting such shifts have utility where the precursor hydrolase is undesirable, e.g. to prevent undesired hydrolysis of a particular substrate in an admixture of substrates. K_(M) and kcat are measured in accord with known procedures, as described in EPO Publication No. 0130756 or as described herein.

Oxidative stability is measured either by known procedures or by the methods described hereinafter. A substantial change in oxidative stability is evidenced by at least about 50% increase or decrease (preferably decrease) in the rate of loss of enzyme activity when exposed to various oxidizing conditions. Such oxidizing conditions are exposure to the organic oxidant diperdodecanoic acid (DPDA) under the conditions described in the examples.

Alkaline stability is measured either by known procedures or by the methods described herein. A substantial change in alkaline stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half life of the enzymatic activity of a mutant when compared to the precursor carbonyl hydrolase. In the case of subtilisins, alkaline stability was measured as a function of autoproteolytic degradation of subtilisin at alkaline pH, e.g. for example, 0.1M sodium phosphate, pH 12 at 25° or 30° C.

Thermal stability is measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half-life of the catalytic activity of a mutant when exposed to a relatively high temperature and neutral pH as compared to the precursor carbonyl hydrolase. In the case of subtilisins, thermal stability is measured by the autoproteolytic degradation of subtilisin at elevated temperatures and neutral pH, e.g., for example 2 mM calcium chloride, 50 mM MOPS pH 7.0 at 59° C.

The inventors have produced mutant subtilisins containing the substitution of the amino acid residues of B. amyloliquefaciens subtilisin shown in Table I. The wild type amino acid sequence and DNA sequence of B. amyloliquefaciens subtilisin is shown in FIG. 1.

                  TABLE I                                                          ______________________________________                                         Residue    Replacement Amino Acid                                              ______________________________________                                         Tyr21      F                                                                   Thr22      C                                                                   Ser24      C                                                                   Asp32      N Q S                                                               Ser33      A T                                                                 Asp36      A G                                                                 Gly46      V                                                                   Ala48      E V R                                                               Ser49      C L                                                                 Met50      C F V                                                               Asn77      D                                                                   Ser87      C                                                                   Lys94      C                                                                   Val95      C                                                                   Tyr104     A C D E F G H I K L M N P Q R S T V W                               Ile107     V                                                                   Gly110     C R                                                                 Met124     I L                                                                 Ala152     G S                                                                 Asn155     A D H Q T                                                           Glu156     Q S                                                                 Gly166     A C D E F H I K L M N P Q R S T V W Y                               Gly169     A C D E F H I K L M N P Q R S T V W Y                               Lys170     E R                                                                 Tyr171     F                                                                   Pro172     E Q                                                                 Phe189     A C D E G H I K L M N P Q R S T V W Y                               Asp197     R A                                                                 Met199     I                                                                   Ser204     C R L P                                                             Lys213     R T                                                                 Tyr217     A C D E F G H I K L M N P Q R S T V W                               Ser221     A C                                                                 Met222     A C D E F G H I K L N P Q R S T V W Y                               ______________________________________                                    

The different amino acids substituted are represented in Table I by the following single letter designations:

                  TABLE XXIII                                                      ______________________________________                                         Amino acid                                                                     or residue       3-letter                                                                               1-letter                                              thereof          symbol  symbol                                                ______________________________________                                         Alanine          Ala     A                                                     Glutamate        Glu     E                                                     Glutamine        Gln     Q                                                     Aspartate        Asp     D                                                     Asparagine       Asn     N                                                     Leucine          Leu     L                                                     Glycine          Gly     G                                                     Lysine           Lys     K                                                     Serine           Ser     S                                                     Valine           Val     V                                                     Arginine         Arg     R                                                     Threonine        Thr     T                                                     Proline          Pro     P                                                     Isoleucine       Ile     I                                                     Methionine       Met     M                                                     Phenylalanine    Phe     F                                                     Tyrosine         Tyr     Y                                                     Cysteine         Cys     C                                                     Tryptophan       Trp     W                                                     Histidine        His     H                                                     ______________________________________                                    

Except where otherwise indicated by context, wild-type amino acids are represented by the above three-letter symbols and replaced amino acids by the above single-letter symbols. Thus, if the methionine at residue 50 in B. amyloliquefaciens subtilisin is replaced by Phenylalanine, this mutation (mutant) may be designated Met50F or F50. Similar designations will be used for multiple mutants.

In addition to the amino acids used to replace the residues disclosed in Table I, other replacements of amino acids at the residues are expected to produce mutant subtilisins having useful properties. These residues and replacement amino acids are shown in Table II.

                  TABLE II                                                         ______________________________________                                         Residue        Replacement Amino Acid(s)                                       ______________________________________                                         Tyr-21         L                                                               Thr22          K                                                               Ser24          A                                                               Asp32                                                                          Ser33          G                                                               Gly46                                                                          Ala48                                                                          Ser49                                                                          Met50          L K I V                                                         Asn77          D                                                               Ser87          N                                                               Lys94          R Q                                                             Val95          L I                                                             Tyr104                                                                         Met124         K A                                                             Ala152         C L I T M                                                       Asn155                                                                         Glu156         A T M L Y                                                       Gly166                                                                         Gly169                                                                         Tyr171         K R E Q                                                         Pro172         D N                                                             Phe189                                                                         Tyr217                                                                         Ser221                                                                         Met222                                                                         ______________________________________                                    

Each of the mutant subtilisins in Table I contain the replacement of a single residue of the B. amyloliquefaciens amino acid sequence. These particular residues were chosen to probe the influence of such substitutions on various properties of B. amyloliquefaciens subtilisin.

Thus, the inventors have identified Met124 and Met222 as important residues which if substituted with another amino acid produce a mutant subtilisin with enhanced oxidative stability. For Met124, Leu and Ile are preferred replacement amino acids. Preferred amino acids for replacement of Met222 are disclosed in EPO Publication No. 0130756.

Various other specific residues have also been identified as being important with regard to substrate specificity. These residues include Tyr104, Ala152, Glu156, Gly166, Gly169, Phe189 and Tyr217 for which mutants containing the various replacement amino acids presented in Table I have already been made, as well as other residues presented below for which mutants have yet to be made.

The identification of these residues, including those yet to be mutated, is based on the inventors' high resolution crystal structure of B. amyloliquefaciens subtilisin to 1.8A (see Table III), their experience with in vitro mutagenesis of subtilisin and the literature on subtilisin. This work and the above referenced x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors, product complexes and transition state analogs has helped in identifying an extended peptide binding cleft in subtilisin. This substrate binding cleft together with substrate is schematically diagrammed in FIG. 2, according to the nomenclature of Schechter, I., et al. (1967) Biochem Bio. Res. Commun. 27, 157. The scissile bond in the substrate is identified by an arrow. The P and P' designations refer to the amino acids which are positioned respectively toward the amino or carboxy terminus relative to the scissile bond. The S and S' designations refer to subsites in the substrate binding cleft of subtilisin which interact with the corresponding substrate amino acid residues.

    ______________________________________                                         1       ALA N    19.434      53.195                                                                               -21.756                                     1       ALA C    18.731      50.985                                                                               -21.324                                     1       ALA CB   21.099      51.518                                                                               -21.183                                     2       GLN CA   17.219      49.008                                                                               -21.434                                     2       GLN D    18.765      47.165                                                                               -21.691                                     2       GLN CG   15.028      47.905                                                                               -21.927                                     2       GLN OE1  13.023      48.612                                                                               -22.867                                     3       SER N    17.477      41.205                                                                               -19.852                                     3       SER C    16.735      44.918                                                                               -19.490                                     3       SER CB   18.588      45.838                                                                               -18.069                                     4       VAL N    16.991      43.646                                                                               -19.725                                     4       VAL C    16.129      41.934                                                                               -18.290                                     4       VAL CB   16.008      41.622                                                                               -20.822                                     4       VAL CG2  16.037      42.266                                                                               -22.186                                     5       PRO CA   15.384      41.415                                                                               -16.027                                     5       PRO O    14.885      39.763                                                                               -17.146                                     5       PRO CG   13.841      43.215                                                                               -15.921                                     6       TYR N    16.363      39.240                                                                               -15.487                                     6       TYR C    15.359      36.975                                                                               -15.528                                     6       TYR CB   17.824      37.323                                                                               -14.834                                     6       TYR CD1  18.437      35.452                                                                               -16.346                                     6       TYR CE1  18.535      34.070                                                                               -16.653                                     6       TYR CZ   18.222      33.154                                                                               -15.628                                     7       GLY K    14.464      37.362                                                                               -14.630                                     7       GLY C    12.400      36.535                                                                               -15.670                                     8       VAL N    12.441      37.529                                                                               -16.541                                     8       VAL C    12.363      36.433                                                                               -18.735                                     8       VAL CB   11.765      38.900                                                                               -18.567                                     8       VAL CG2  10.991      39.919                                                                               -17.733                                     9       SER CA   14.419      35.342                                                                               -19.562                                     9       SER O    14.112      33.014                                                                               -19.801                                     9       SER OG   16.162      36.747                                                                               -20.358                                     10      GLN CA   13.964      32.636                                                                               -16.876                                     10      GLN O    12.785      30.642                                                                               -17.413                                     10      GLN CG   14.295      31.617                                                                               -14.588                                     10      GLN OE1  14.554      33.068                                                                               -12.744                                     11      ILE N    11.625      32.575                                                                               -17.670                                     11      ILE C    10.209      31.792                                                                               -19.605                                     11      ILE CB   9.132       32.669                                                                               -17.475                                     11      ILE CGZ  9.162       32.655                                                                               -15.941                                     11      IYS N    11.272      32.185                                                                               -20.277                                     12      LYS C    10.456      33.006                                                                               -22.522                                     12      LYS CB   11.257      30.646                                                                               -22.216                                     12      LYS CD   12.543      28.517                                                                               -22.159                                     12      LYS NZ   14.476      27.680                                                                               -20.935                                     13      ALA CA   9.325       35.198                                                                               -22.631                                     13      ALA O    9.338       35.804                                                                               -24.901                                     14      PRO N    11.332      35.950                                                                               -23.893                                     14      PRO C    11.786      35.557                                                                               -26.317                                     14      PRO CB   13.462      36.580                                                                               -24.692                                     14      PRO CD   12.282      35.936                                                                               -22.758                                     15      ALA CA   11.379      33.450                                                                               -27.367                                     15      ALA O    10.008      33.710                                                                               -29.278                                     16      LEU N    9.085       34.138                                                                               -27.240                                     16      LEU C    7.912       35.925                                                                               -28.521                                     16      LEU CB   6.746       34.623                                                                               -26.698                                     16      LEU CD1  5.001       33.234                                                                               -27.809                                     17      HIS N    8.665       36.828                                                                               -27.922                                     17      HIS C    9.510       37.981                                                                               -29.890                                     17      HIS CB   9.708       39.100                                                                               -27.652                                     17      HIS ND1  9.930       39.887                                                                               -25.272                                     17      HIS CE1  9.226       39.914                                                                               -24.144                                     18      SER N    10.443      37.033                                                                               -30.022                                     1       ALA CA   19.811      51.774                                                                               -21.965                                     1       ALA O    18.376      51.197                                                                               -20.175                                     2       GLN N    18.268      49.886                                                                               -22.041                                     2       GLN C    17.875      47.706                                                                               -20.992                                     2       GLN CB   16.125      48.760                                                                               -22.449                                     2       GLN CD   13.912      47.762                                                                               -22.930                                     2       GLN NE2  14.115      46.917                                                                               -23.926                                     3       SER CA   17.950      45.868                                                                               -19.437                                     3       SER O    15.590      45.352                                                                               -19.229                                     3       SER OG   17.682      46.210                                                                               -17.049                                     4       VAL CA   15.946      42.619                                                                               -19.639                                     4       VAL O    17.123      41.178                                                                               -18.086                                     4       VAL CG1  14.874      40.572                                                                               -20.741                                     5       PRO N    15.239      42.106                                                                               -17.331                                     5       PRO C    15.501      39.905                                                                               -16.249                                     5       PRO CB   14.150      41.880                                                                               -15.263                                     5       PRO CD   14.044      42.986                                                                               -17.417                                     6       TYR CA   16.628      37.803                                                                               -15.715                                     6       TYR D    15.224      35.943                                                                               -16.235                                     6       TYR CG   18.021      35.847                                                                               -15.055                                     6       TYR CD2  17.696      34.908                                                                               -14.071                                     6       TYR CE2  17.815      33.539                                                                               -14.379                                     6       TYR OH   18.312      31.838                                                                               -15.996                                     7       GLY CA   13.211      36.640                                                                               -14.376                                     7       GLY O    11.747      35.478                                                                               -15.883                                     8       VAL CA   11.777      37.523                                                                               -17.836                                     8       VAL D    11.639      35.716                                                                               -19.470                                     8       VAL CG1  11.106      38.893                                                                               -19.943                                     9       SER N    13.661      36.318                                                                               -18.775                                     9       SER C    14.188      33.920                                                                               -18.965                                     9       SER CB   15.926      35.632                                                                               -19.505                                     10      GLN N    14.115      33.887                                                                               -17.662                                     10      GLN C    12.687      31.887                                                                               -17.277                                     10      GLN CB   14.125      32.885                                                                               -15.410                                     10      GLN CD   14.486      31.911                                                                               -13.147                                     10      GLN NE2  14.552      30.960                                                                               -12.251                                     11      ILE CA   10.373      31.904                                                                               -18.102                                     11      ILE O    9.173       31.333                                                                               -20.180                                     11      ILE CG1  9.066       34.117                                                                               -18.049                                     11      ILE CD1  7.588       34.648                                                                               -17.923                                     12      LYS CA   11.388      32.119                                                                               -21.722                                     12      LYS O    10.173      32.703                                                                               -23.686                                     12      LYS CG   12.283      29.830                                                                               -21.423                                     12      LYS CE   13.023      27.467                                                                               -21.166                                     13      ALA N    10.109      34.138                                                                               -21.991                                     13      ALA C    10.026      35.716                                                                               -23.863                                     13      ALA CB   8.885       36.195                                                                               -21.565                                     14      PRO CA   11.985      36.430                                                                               -25.720                                     14      PRO O    11.778      36.047                                                                               -27.445                                     14      PRO CG   13.328      36.978                                                                               -23.221                                     15      ALA N    11.560      34.236                                                                               -26.129                                     15      ALA C    10.082      33.795                                                                               -28.032                                     15      ALA CB   11.552      31.969                                                                               -27.062                                     16      LEU CA   7.791       34.558                                                                               -27.828                                     16      LEU O    7.342       36.126                                                                               -29.588                                     16      LEU CG   5.790       33.465                                                                               -26.522                                     16      LEU CD2  6.694       32.287                                                                               -26.283                                     17      HIS CA   8.890       38.151                                                                               -28.530                                     17      HIS O    9.107       38.622                                                                               -30.856                                     17      HIS CG   9.185       39.288                                                                               -26.262                                     17      HIS CD2  8.008       38.924                                                                               -25.694                                     17      HIS NE2  8.079       39.328                                                                               -24.381                                     18      SER CA   11.109      36.739                                                                               -31.322                                     18      SER C    10.159      36.123                                                                               -32.353                                     18      SER CB   12.311      35.799                                                                               -31.172                                     19      GLN N    9.080       35.485                                                                               -31.943                                     19      GLN C    7.142       36.111                                                                               -33.303                                     19      GLN CB   7.221       33.849                                                                               -32.280                                     19      GLN CD   6.923       31.707                                                                               -31.181                                     19      GLN NE2  7.362       30.852                                                                               -30.256                                     20      GLY CA   6.369       38.387                                                                               -32.859                                     20      GLY D    4.263       39.276                                                                               -32.215                                     21      TYR CA   4.118       37.831                                                                               -29.763                                     21      TYR D    5.422       38.074                                                                               -27.756                                     21      TYR CG   2.973       35.784                                                                               -30.708                                     21      TYR CD2  3.650       34.794                                                                               -31.397                                     21      TYR CE2  3.193       34.261                                                                               -32.588                                     21      TYR OH   1.501       34.241                                                                               -34.250                                     22      THR CA   4.262       40.527                                                                               -27.129                                     22      THR O    3.287       41.725                                                                               -25.325                                     22      THR DG1  4.319       42.457                                                                               -28.597                                     23      GLY N    1.939       40.285                                                                               -26.453                                     23      GLY C    -0.157      41.631                                                                               -26.118                                     24      SER N    -0.023      41.967                                                                               -27.371                                     24      SER C    -2.383      42.626                                                                               -27.864                                     24      SER CB   -0.734      43.120                                                                               -29.520                                     25      ASN N    -3.059      43.692                                                                               -27.515                                     25      ASN C    -5.015      42.875                                                                               -26.205                                     25      ASN CB   -5.165      43.227                                                                               -28.700                                     25      ASN OD1  -4.965      43.767                                                                               -31.083                                     26      VAL N    -4.177      42.449                                                                               -25.292                                     26      VAL C    -4.792      42.652                                                                               -22.987                                     26      VAL CB   -3.714      40.503                                                                               -23.821                                     26      VAL CG2  -3.598      39.576                                                                               -25.018                                     27      LYS CA   -6.133      43.524                                                                               -21.175                                     27      LYS O    -6.405      41.873                                                                               -19.413                                     27      LYS CG   -8.046      44.575                                                                               -22.490                                     27      LYS CE   -10.304     45.497                                                                               -23.137                                     28      VAL N    -4.818      43.462                                                                               -19.200                                     28      VAL C    -4.758      43.959                                                                               -16.828                                     28      VAL CB   -2.926      42.666                                                                               -17.932                                     28      VAL CG2  -2.667      41.805                                                                               -19.173                                     29      ALA CA   -5.747      44.330                                                                               -14.639                                     29      ALA D    -4.666      42.845                                                                               -13.104                                     30      VAL N    -4.057      45.033                                                                               -13.072                                     30      VAL C    -3.958      45.409                                                                               -10.681                                     30      VAL CB   -1.886      45.810                                                                               -12.149                                     30      VAL CG2  -1.053      45.236                                                                               -13.307                                     31      ILE CA   -5.328      44.846                                                                               -8.679                                      31      ILE D    -3.825      43.915                                                                               -6.997                                      31      ILE CG1  -7.298      43.707                                                                               -9.798                                      31      ILE CD1  -8.617      42.856                                                                               -9.717                                      32      ASP CA   -2.944      46.467                                                                               -6.255                                      32      ASP D    -4.197      48.418                                                                               -5.502                                      32      ASP CG   -0.483      45.702                                                                               -6.273                                      32      ASP OD2  -0.081      46.429                                                                               -5.330                                      33      SER CA   -1.895      49.857                                                                               -4.801                                      33      SER D    -1.706      52.136                                                                               -5.363                                      33      SER OG   0.535       50.025                                                                               -4.774                                      34      GLY CA   -2.255      51.728                                                                               -8.165                                      34      GLY D    -0.144      50.831                                                                               -8.761                                      35      ILE CA   0.208       52.438                                                                               -10.995                                     35      ILE D    -0.327      54.638                                                                               -11.744                                     35      ILE CG1  -0.530      50.210                                                                               -12.097                                     35      ILE CD1  -0.962      49.485                                                                               -13.424                                     36      ASP CA   2.359       55.618                                                                               -11.232                                     18      SER O    10.547      36.112                                                                               -33.534                                     18      SER OG   13.321      36.450                                                                               -30.399                                     19      GLN CA   8.082       34.962                                                                               -32.878                                     19      GLN D    6.297       35.972                                                                               -34.219                                     19      GLN CG   7.975       32.602                                                                               -31.823                                     19      GLN DE1  5.719       31.833                                                                               -31.444                                     20      GLY N    7.205       37.223                                                                               -32.587                                     20      GLY C    5.181       38.492                                                                               -31.880                                     21      TYR N    5.202       37.801                                                                               -30.761                                     21      TYR C    4.579       38.552                                                                               -28.525                                     21      TYR CB   3.498       36.431                                                                               -29.443                                     21      TYR CD1  1.795       36.332                                                                               -31.258                                     21      TYR CE1  1.306       35.797                                                                               -32.446                                     21      TYR CZ   2.003       34.755                                                                               -33.067                                     22      THR N    3.902       39.680                                                                               -28.288                                     22      THR C    3.091       40.922                                                                               -26.244                                     22      THR CB   5.133       41.759                                                                               -27.611                                     22      THR CG2  6.476       41.323                                                                               -28.229                                     23      GLY CA   0.809       40.600                                                                               -25.542                                     23      GLY D    -1.013      42.095                                                                               -25.330                                     24      SER CA   -0.897      42.957                                                                               -28.012                                     24      SER D    -2.813      41.508                                                                               -28.160                                     24      SER OG   0.563       43.652                                                                               -29.728                                     25      ASN CA   -4.519      43.687                                                                               -27.393                                     25      ASN O    -6.233      42.668                                                                               -26.190                                     25      ASN CG   -4.960      44.170                                                                               -29.885                                     25      ASN NO2  -4.747      45.461                                                                               -29.594                                     26      VAL CA   -4.674      41.679                                                                               -24.143                                     26      VAL D    -3.858      43.419                                                                               -22.689                                     26      VAL CG1  -4.160      39.802                                                                               -22.548                                     27      LYS N    -5.910      42.613                                                                               -22.301                                     27      LYS C    -5.815      42.872                                                                               -19.841                                     27      LYS CB   -7.590      43.981                                                                               -21.149                                     27      LYS CD   -9.321      45.302                                                                               -22.020                                     27      LYS NZ   -9.686      46.253                                                                               -24.264                                     28      VAL CA   -4.457      42.950                                                                               -17.897                                     28      VAL O    -4.209      45.095                                                                               -16.817                                     28      VAL CG1  -2.466      42.105                                                                               -16.589                                     29      ALA N    -5.484      43.527                                                                               -15.813                                     29      ALA C    -4.750      44.010                                                                               -13.553                                     29      ALA CB   -7.172      44.107                                                                               -14.101                                     30      VAL CA   -3.146      44.962                                                                               -11.910                                     30      VAL D    -4.155      46.648                                                                               -10.578                                     30      VAL CG1  -0.996      45.901                                                                               -10.900                                     31      ILE N    -4.514      44.515                                                                               -9.877                                      31      ILE C    -4.346      44.933                                                                               -7.546                                      31      ILE CB   -6.457      43.776                                                                               -8.501                                      31      ILE CG2  -7.278      44.038                                                                               -7.225                                      32      ASP N    -4.044      46.193                                                                               -7.227                                      32      ASP C    -3.071      47.889                                                                               -5.705                                      32      ASP CB   -1.695      46.129                                                                               -7.092                                      32      ASP OD1  0.034       44.592                                                                               -6.576                                      33      SER N    -1.931      48.512                                                                               -5.394                                      33      SER C    -1.952      50.976                                                                               -5.808                                      33      SER CB   -0.621      49.922                                                                               -3.939                                      34      GLY N    -2.173      50.740                                                                               -7.084                                      34      GLY C    -1.035      51.648                                                                               -9.057                                      35      ILE N    -0.965      52.431                                                                               -10.102                                     35      ILE C    0.568       53.919                                                                               -11.263                                     35      ILE CB   -0.042      51.694                                                                               -12.367                                     35      ILE CG2  1.149       51.741                                                                               -13.362                                     36      ASP N    1.816       54.253                                                                               -10.971                                     36      ASP C    2.281       55.956                                                                               -12.702                                     36      ASP D    3.004       55.471                                                                               -13.579                                     36      ASP CG   4.339       51.099                                                                               -10.804                                     36      ASP OD2  5.448       57.277                                                                               -10.263                                     37      SER CA   1.183       51.221                                                                               -14.512                                     37      SER O    2.545       58.303                                                                               -16.151                                     37      SER OG   -0.090      59.133                                                                               -13.879                                     38      SER CA   4.261       59.505                                                                               -14.487                                     38      SER D    6.543       59.251                                                                               -15.285                                     38      SER DG   5.376       59.865                                                                               -12.234                                     39      HIS CA   6.637       56.574                                                                               -15.291                                     39      HIS D    5.738       55.878                                                                               -17.419                                     39      HIS CG   8.014       54.609                                                                               -14.456                                     39      HIS CD2  8.769       54.345                                                                               -13.389                                     39      HIS NE2  9.986       53.910                                                                               -13.808                                     40      PRO CA   7.988       56.697                                                                               -18.831                                     40      PRO D    8.032       55.097                                                                               -20.578                                     40      PRO CG   10.053      57.405                                                                               -17.902                                     41      ASP N    8.481       54.328                                                                               -18.485                                     41      ASP OD1  10.325      51.395                                                                               -20.429                                     41      ASP CB   9.799       52.239                                                                               -18.224                                     41      ASP C    7.311       52.163                                                                               -18.839                                     42      LEU N    6.185       52.803                                                                               -18.558                                     42      LEU C    3.924       52.907                                                                               -19.376                                     42      LEU CB   4.421       52.158                                                                               -17.008                                     42      LEU CD1  4.535       51.546                                                                               -14.581                                     43      LYS N    3.018       52.135                                                                               -19.946                                     43      LYS C    0.637       52.156                                                                               -20.018                                     43      LYS CB   2.021       52.389                                                                               -22.169                                     43      LYS CD   0.998       52.862                                                                               -24.339                                     43      LYS NZ   0.337       51.757                                                                               -26.418                                     44      VAL CA   -1.407      52.639                                                                               -18.765                                     44      VAL O    -2.623      53.906                                                                               -20.434                                     44      VAL CG1  -2.724      52.941                                                                               -16.582                                     45      ALA N    -3.494      51.951                                                                               -19.871                                     45      ALA C    -5.841      52.507                                                                               -20.053                                     45      ALA CB   -4.831      50.580                                                                               -21.389                                     46      GLY CA   -7.082      52.837                                                                               -18.001                                     46      GLY O    -5.938      52.006                                                                               -16.035                                     47      GLY CA   -8.014      52.246                                                                               -14.388                                     47      GLY O    -9.988      53.481                                                                               -14.185                                     48      ALA CA   -10.255     52.870                                                                               -11.382                                     48      ALA D    -9.066      51.720                                                                               -9.725                                      49      SER N    -10.149     53.547                                                                               -9.037                                      49      SER C    -10.947     52.986                                                                               -6.783                                      49      SER CB   -9.092      54.588                                                                               -7.029                                      50      MET N    -10.835     52.007                                                                               -5.932                                      50      MET C    -11.463     51.962                                                                               -3.561                                      50      MET CB   -12.012     50.018                                                                               -4.996                                      50      MET SD   -13.460     49.889                                                                               -7.256                                      51      VAL N    -10.427     52.760                                                                               -3.422                                      51      VAL C    -10.630     54.562                                                                               -1.907                                      51      VAL CB   -8.443      53.155                                                                               -2.000                                      51      VAL CG2  -7.764      51.815                                                                               -2.302                                      52      PRO CA   -12.372     55.933                                                                               -0.821                                      52      PRO D    -11.771     58.220                                                                               -0.925                                      52      PRO CG   -13.583     54.103                                                                               0.085                                       53      SER N    -10.442     56.906                                                                               0.299                                       53      SER C    -8.420      58.245                                                                               -0.326                                      53      SER CB   -9.004      57.707                                                                               2.069                                       54      GLU N    -8.254      57.523                                                                               -1.393                                      54      GLU C    -7.767      57.303                                                                               -3.785                                      54      GLU CB   -6.134      56.599                                                                               -2.154                                      54      GLU CD   -4.066      56.062                                                                               -0.970                                      36      ASP CB   3.712       55.720                                                                               -10.514                                     36      ASP OD1  3.755       57.974                                                                               -11.429                                     37      SER N    1.304       56.822                                                                               -13.111                                     37      SER C    2.377       58.095                                                                               -14.949                                     37      SER CB   -0.093      58.049                                                                               -14.788                                     38      SER N    3.163       58.614                                                                               -14.001                                     38      SER C    5.466       58.705                                                                               -14.992                                     38      SER CB   4.742       60.435                                                                               -13.398                                     39      HIS N    5.454       57.390                                                                               -14.892                                     39      HIS C    6.681       56.401                                                                               -16.778                                     39      HIS CB   6.637       55.203                                                                               -14.515                                     39      HIS ND1  8.795       54.356                                                                               -15.561                                     39      HIS CE1  9.970       53.930                                                                               -15.130                                     40      PRO N    7.807       56.836                                                                               -17.387                                     40      PRO C    8.156       55.280                                                                               -19.357                                     40      PR0 CB   9.247       57.533                                                                               -19.161                                     40      PRO CD   8.988       57.452                                                                               -16.776                                     41      ASP OD2  11.148      50.399                                                                               -18.668                                     41      ASP CG   10.473      51.307                                                                               -19.211                                     41      ASP CA   8.645       52.959                                                                               -18.966                                     41      ASP O    7.396       50.947                                                                               -18.977                                     42      LEU CA   4.892       52.147                                                                               -18.466                                     42      LEU D    3.993       54.163                                                                               -19.490                                     42      LEU CG   5.182       51.363                                                                               -15.946                                     42      LEU CD2  5.273       49.877                                                                               -16.350                                     43      LYS CA   1.893       52.685                                                                               -20.721                                     43      LYS O    0.504       50.920                                                                               -19.820                                     43      LYS CG   0.685       52.436                                                                               -22.910                                     43      LYS CE   -0.180      52.584                                                                               -25.260                                     44      VAL N    -0.191      53.035                                                                               -19.490                                     44      VAL C    -2.571      52.887                                                                               -19.731                                     44      VAL CB   -1.480      53.351                                                                               -11.383                                     44      VAL CG2  -0.197      53.194                                                                               -16.553                                     45      ALA CA   -4.619      51.977                                                                               -20.810                                     45      ALA O    -6.703      53.085                                                                               -20.703                                     46      GLY N    -5.910      52.356                                                                               -18.768                                     46      GLY C    -6.987      52.443                                                                               -16.538                                     47      GLY N    -8.092      52.658                                                                               -15.793                                     47      GLY C    -9.179      52.757                                                                               -13.572                                     48      ALA N    -9.221      52.446                                                                               -12.330                                     48      ALA C    -9.790      52.675                                                                               -9.968                                      48      ALA CB   -11.558     52.100                                                                               -11.617                                     49      SER CA   -9.752      53.355                                                                               -7.652                                      49      SER O    -11.972     53.677                                                                               -6.908                                      49      SER OG   -8.879      54.255                                                                               -5.650                                      50      MET CA   -11.852     51.549                                                                               -4.974                                      50      MET O    -11.997     51.398                                                                               -2.575                                      50      MET CG   -11.912     49.463                                                                               -6.389                                      50      MET CE   -12.808     50.111                                                                               -8.903                                      51      VAL CA   -9.968      53.170                                                                               -2.067                                      51      VAL O    -10.237     55.437                                                                               -2.682                                      51      VAL CG1  -7.892      53.579                                                                               -0.631                                      52      PRO N    -11.621     54.693                                                                               -1.056                                      52      PRO C    -11.490     57.123                                                                               -0.440                                      52      PRO CB   -13.400     55.594                                                                               0.244                                       52      PRO CD   -12.164     53.620                                                                               -0.175                                      53      SER CA   -9.538      57.982                                                                               0.682                                       53      SER O    -7.679      59.224                                                                               -0.038                                      53      SER OG   -8.256      56.521                                                                               2.127                                       54      GLU CA   -7.204      57.648                                                                               -2.421                                      54      GLU O    -7.533      56.243                                                                               -4.379                                      54      GLU CG   -5.289      56.959                                                                               -0.927                                      54      GLU OE1  -3.545      55.694                                                                               -1.968                                      54      GLU OE2  -3.900      55.777                                                                               0.271                                       55      THR CA   -9.433      58.121                                                                               -5.441                                      55      THR D    -9.433      51.919                                                                               -7.810                                      55      THR OG1  -9.885      60.510                                                                               -5.418                                      56      ASN N    -7.482      58.403                                                                               -6.877                                      56      ASN OD1  -5.075      58.967                                                                               -10.337                                     56      ASN CB   -5.898      59.694                                                                               -8.208                                      56      ASN C    -6.012      57.094                                                                               -8.305                                      57      PRO N    -6.362      56.261                                                                               -9.258                                      57      PRO CD   -7.384      56.433                                                                               -10.272                                     57      PRO CA   -5.679      54.961                                                                               -9.332                                      57      PRO O    -3.509      54.128                                                                               -9.945                                      58      PHE CA   -2.747      56.577                                                                               -11.222                                     58      PHE O    -0.635      57.497                                                                               -10.680                                     58      PHE CG   -3.983      56.968                                                                               -13.357                                     58      PHE CD2  -5.211      57.630                                                                               -13.459                                     58      PHE CE2  -6.194      57.095                                                                               -14.276                                     59      GLN N    -2.044      57.119                                                                               -8.990                                      59      GLN C    -0.807      56.403                                                                               -7.000                                      59      GLN CB   -1.862      58.668                                                                               -7.089                                      59      GLN CD   -1.790      60.157                                                                               -5.150                                      59      GLN NE2  -2.959      59.685                                                                               -4.742                                      60      ASP CA   0.851       54.792                                                                               -6.304                                      60      ASP D    2.827       55.550                                                                               -5.231                                      60      ASP CG   2.077       52.538                                                                               -6.380                                      60      ASP OD2  2.915       51.841                                                                               -7.030                                      61      ASN ND2  -1.364      57.747                                                                               -2.347                                      61      ASN CG   -0.040      51.670                                                                               -2.399                                      61      ASN CA   1.557       55.734                                                                               -2.700                                      61      ASN O    2.933       54.862                                                                               -0.902                                      62      ASN CA   2.877       52.348                                                                               -1.709                                      62      ASN D    4.951       51.313                                                                               -1.770                                      62      ASN CG   2.371       50.103                                                                               -0.697                                      62      ASN ND2  2.622       50.208                                                                               0.601                                       63      SER CA   5.189       51.696                                                                               -4.709                                      63      SER O    5.593       49.790                                                                               -6.269                                      63      SER OG   6.871       50.698                                                                               -3.418                                      64      HIS CA   3.994       48.059                                                                               -4.935                                      64      HIS O    3.861       46.974                                                                               -7.108                                      64      HIS CG   3.144       46.021                                                                               -3.726                                      64      HIS CD2  4.054       45.194                                                                               -3.135                                      64      HIS NE2  3.556       43.920                                                                               -3.368                                      65      GLY CA   1.552       48.264                                                                               -7.830                                      65      GLY O    2.230       48.078                                                                               -10.134                                     66      THR CA   4.064       50.117                                                                               -9.954                                      66      THR D    5.333       48.789                                                                               -11.461                                     66      THR OG1  3.637       52.425                                                                               -9.406                                      67      HIS N    5.685       48.443                                                                               -9.274                                      67      HIS C    6.091       46.141                                                                               -10.143                                     67      HIS CB   7.300       47.071                                                                               -8.064                                      67      HIS ND1  8.590       44.907                                                                               -8.276                                      67      HIS CE1  9.857       44.491                                                                               -8.299                                      68      VAL N    4.892       45.749                                                                               -9.731                                      68      VAL C    3.856       44.860                                                                               -11.740                                     68      VAL CB   2.939       44.252                                                                               -9.386                                      68      VAL CG2  3.319       43.705                                                                               -8.000                                      69      ALA CA   3.037       46.468                                                                               -13.429                                     69      ALA D    4.028       45.913                                                                               -15.565                                     70      GLY N    5.340       46.782                                                                               -13.914                                     70      GLY C    7.046       45.370                                                                               -15.021                                     71      THR N    6.820       44.431                                                                               -14.138                                     71      THR C    6.224       42.506                                                                               -15.543                                     71      THR CB   7.119       42.070                                                                               -13.191                                     55      THR N    -8.571      58.251                                                                               -4.249                                      55      THR C    -8.764      58.139                                                                               -6.779                                      55      THR CB   -10.586     59.200                                                                               -5.303                                      55      THR CG2  -11.432     59.143                                                                               -4.017                                      56      ASN ND2  -4.930      61.179                                                                               -9.881                                      56      ASN CG   -5.273      59.925                                                                               -9.555                                      56      ASN CA   -6.762      58.425                                                                               -8.200                                      56      ASN D    -5.104      56.866                                                                               -7.470                                      57      PRO CG   -7.123      55.257                                                                               -11.177                                     57      PRO CB   -6.644      54.178                                                                               -10.235                                     57      PRO C    -4.301      55.082                                                                               -9.966                                      58      PHE N    -3.998      56.262                                                                               -10.491                                     58      PHE C    -1.712      57.129                                                                               -10.253                                     58      PHE CB   -2.943      57.502                                                                               -12.423                                     58      PHE CD1  -3.756      55.780                                                                               -14.059                                     58      PHE CE1  -4.722      55.255                                                                               -14.928                                     58      PHE CZ   -5.949      55.939                                                                               -15.051                                     59      GLN CA   -1.172      57.583                                                                               -7.934                                      59      GLN D    -1.639      56.083                                                                               -6.115                                      59      GLN CG   -0.942      59.261                                                                               -6.034                                      59      GLN OE1  -1.404      61.288                                                                               -4.836                                      60      ASP N    0.410       55.895                                                                               -7.211                                      60      ASP C    1.631       55.267                                                                               -5.090                                      60      ASP CB   1.596       53.744                                                                               -7.188                                      60      ASP OD1  1.746       52.337                                                                               -5.190                                      61      ASN N    0.959       55.265                                                                               -3.950                                      61      ASN OD1  0.666       58.566                                                                               -2.875                                      61      ASN CB   0.531       56.401                                                                               -1.784                                      61      ASN C    2.291       54.632                                                                               -1.940                                      62      ASN N    2.210       53.434                                                                               -2.468                                      62      ASN C    4.124       51.893                                                                               -2.479                                      62      ASN CB   1.783       51.319                                                                               -1.421                                      62      ASN OD1  2.633       49.077                                                                               -1.343                                      63      SER N    4.152       52.104                                                                               -3.761                                      63      SER C    5.071       50.256                                                                               -5.209                                      63      SER CB   6.523       51.958                                                                               -4.012                                      64      HIS N    4.202       49.475                                                                               -4.639                                      64      HIS C    3.366       47.759                                                                               -6.261                                      64      HIS CB   3.184       47.501                                                                               -3.747                                      64      HIS ND1  2.107       45.247                                                                               -4.241                                      64      HIS CE1  2.416       43.966                                                                               -4.054                                      65      GLY N    2.287       48.428                                                                               -6.587                                      65      GLY C    2.392       48.636                                                                               -9.037                                      66      THR N    3.233       49.659                                                                               -8.832                                      66      THR C    5.089       49.009                                                                               -10.291                                     66      THR CB   4.744       51.511                                                                               -9.667                                      66      THR CG2  5.536       52.078                                                                               -10.849                                     67      HIS CA   6.703       47.361                                                                               -9.458                                      67      HIS O    6.649       45.638                                                                               -11.150                                     67      HIS CG   8.595       46.275                                                                               -8.148                                      67      HIS CD2  9.904       46.678                                                                               -8.076                                      67      HIS N52  10.678      45.514                                                                               -8.186                                      68      VAL CA   4.142       44.607                                                                               -10.266                                     68      VAL O    4.114       43.942                                                                               -12.535                                     68      VAL CG1  1.960       43.260                                                                               -10.020                                     69      ALA N    3.373       46.049                                                                               -12.113                                     69      ALA C    4.193       46.390                                                                               -14.411                                     69      ALA CB   2.332       47.851                                                                               -13.386                                     70      GLY CA   6.595       46.805                                                                               -14.670                                     70      GLY O    7.604       45.154                                                                               -16.119                                     71      THR CA   7.177       43.019                                                                               -14.446                                     71      THR O    6.602       41.828                                                                               -16.495                                     71      THR OG1  8.191       42.592                                                                               -12.390                                     71      THR CG2  7.274       40.583                                                                               -13.596                                     72      VAL CA   3.976       42.491                                                                               -16.484                                     72      VAL D    4.341       42.380                                                                               -18.860                                     72      VAL CG1  1.512       42.480                                                                               -17.170                                     73      ALA N    4.504       44.417                                                                               -17.880                                     73      ALA C    5.433       46.333                                                                               -19.355                                     73      ALA CB   3.107       45.441                                                                               -19.433                                     74      ALA CA   7.470       47.591                                                                               -18.859                                     74      ALA O    7.959       46.640                                                                               -21.054                                     75      LEU N    7.650       48.784                                                                               -21.039                                     75      LEU C    9.192       48.568                                                                               -22.966                                     75      LEU CB   7.548       50.471                                                                               -22.809                                     75      LEU CD1  6.079       52.436                                                                               -22.300                                     76      ASN N    9.147       46.103                                                                               -24.169                                     76      ASN OD1  10.950      45.840                                                                               -27.928                                     76      ASN CB   10.010      46.651                                                                               -25.908                                     76      ASN C    10.783      49.048                                                                               -25.643                                     77      ASN N    11.804      49.664                                                                               -25.071                                     77      ASN C    13.707      51.029                                                                               -25.348                                     77      ASN CB   11.335      52.076                                                                               -25.117                                     77      ASN OD1  12.032      51.346                                                                               -22.917                                     78      SER N    14.125      52.267                                                                               -25.164                                     78      SER C    15.810      52.742                                                                               -23.436                                     78      SER CB   15.905      53.941                                                                               -25.587                                     79      ILE N    14.858      52.565                                                                               -22.529                                     79      ILE C    14.617      51.683                                                                               -20.230                                     79      ILE CB   14.471      54.174                                                                               -20.697                                     79      ILE CG2  14.997      55.320                                                                               -21.612                                     80      GLY N    14.995      51.768                                                                               -18.981                                     80      GLY C    14.612      49.448                                                                               -18.219                                     81      VAL N    13.513      48.766                                                                               -17.980                                     81      VAL C    12.511      46.919                                                                               -19.217                                     81      VAL CB   13.001      46.755                                                                               -16.677                                     81      VAL CG2  11.638      47.261                                                                               -16.231                                     82      LEU CA   11.312      45.020                                                                               -20.256                                     82      LEU O    10.858      43.356                                                                               -18.600                                     82      LEU CG   11.430      43.568                                                                               -22.366                                     82      LEU CD2  12.359      42.675                                                                               -23.192                                     83      GLY CA   8.133       43.321                                                                               -19.114                                     83      GLY D    8.546       41.822                                                                               -21.026                                     84      VAL CA   6.973       39.807                                                                               -19.888                                     84      VAL O    6.424       39.472                                                                               -22.194                                     84      VAL CG1  5.680       37.677                                                                               -19.557                                     85      ALA N    5.156       40.926                                                                               -21.024                                     85      ALA C    4.213       42.683                                                                               -22.396                                     85      ALA CB   2.846       40.663                                                                               -21.748                                     86      PRO CA   5.413       44.635                                                                               -23.205                                     86      PRO O    4.291       46.605                                                                               -23.849                                     86      PRO CG   7.030       43.468                                                                               -24.546                                     87      SER N    3.548       44.676                                                                               -24.769                                     87      SER C    1.103       45.132                                                                               -24.897                                     87      SER CB   2.401       44.777                                                                               -26.927                                     88      ALA N    1.017       44.564                                                                               -23.742                                     88      ALA CA   -0.273      44.353                                                                               -23.084                                     88      ALA O    -0.174      46.717                                                                               -22.435                                     89      SER OG   -4.146      47.102                                                                               -24.280                                     89      SER CA   -3.001      46.867                                                                               -22.227                                     89      SER O    3.793       45.864                                                                               -20.209                                     90      LEU CA   -2.378      47.667                                                                               -18.593                                     90      LEU D    -3.582      49.604                                                                               -18.215                                     90      LEU CG   -0.233      47.851                                                                               -17.174                                     90      LEU CD2  1.160       48.524                                                                               -17.047                                     91      TYR CA   -5.258      48.678                                                                               -16.137                                     72      VAL N    4.930       42.887                                                                               -15.427                                     72      VAL C    4.312       43.084                                                                               -17.831                                     72      VAL CB   2.516       42.867                                                                               -16.085                                     72      VAL CG2  2.142       42.327                                                                               -14.723                                     73      ALA CA   4.587       45.091                                                                               -19.167                                     73      ALA D    5.062       47.188                                                                               -20.216                                     74      ALA N    6.544       46.429                                                                               -18.635                                     74      ALA C    7.740       47.648                                                                               -20.342                                     74      ALA CB   8.653       47.446                                                                               -17.925                                     75      LEU CA   7.812       48.968                                                                               -22.456                                     75      LEU O    10.162      48.750                                                                               -22.253                                     75      LEU CG   6.123       50.913                                                                               -22.379                                     75      LEU CD2  5.096       50.442                                                                               -23.405                                     76      ASN ND2  12.385      46.432                                                                               -26.304                                     76      ASN CG   11.195      46.274                                                                               -26.802                                     76      ASN CA   10.359      47.738                                                                               -24.938                                     76      ASN O    10.157      49.479                                                                               -26.619                                     77      ASN CA   12.220      50.957                                                                               -25.681                                     77      ASN O    14.364      49.979                                                                               -25.313                                     77      ASN CG   11.250      52.027                                                                               -23.616                                     77      ASN ND2  10.294      52.741                                                                               -23.025                                     78      SER CA   15.513      52.614                                                                               -24.906                                     78      SER D    16.982      53.071                                                                               -23.164                                     78      SER OG   15.926      53.870                                                                               -26.999                                     79      ILE CA   15.155      52.784                                                                               -21.120                                     79      ILE D    13.843      50.841                                                                               -20.679                                     79      ILE CG1  12.945      54.032                                                                               -20.814                                     79      ILE CD1  12.135      55.176                                                                               -20.155                                     80      GLY CA   14.476      50.940                                                                               -17.913                                     80      GLY D    15.719      48.994                                                                               -18.544                                     81      VAL CA   13.411      47.286                                                                               -18.061                                     81      VAL D    12.260      47.739                                                                               -20.117                                     81      VAL CG1  14.030      47.084                                                                               -15.573                                     82      LEU N    12.126      45.645                                                                               -19.216                                     82      LEU C    10.390      44.028                                                                               -19.510                                     82      LEU CB   12.206      44.219                                                                               -21.229                                     82      LEU CD1  10.796      44.657                                                                               -23.223                                     83      GLY N    9.131       44.180                                                                               -19.816                                     83      GLY C    8.027       42.011                                                                               -19.925                                     84      VAL N    7.272       41.112                                                                               -19.283                                     84      VAL C    6.164       40.030                                                                               -21.140                                     84      VAL CB   6.256       38.920                                                                               -18.841                                     84      VAL CG2  7.190       38.507                                                                               -17.705                                     85      ALA CA   4.217       41.194                                                                               -22.158                                     85      ALA D    3.260       43.401                                                                               -22.030                                     86      PRO N    5.240       43.186                                                                               -23.059                                     86      PRO C    4.321       45.371                                                                               -23.947                                     86      PRO CB   6.822       44.784                                                                               23.813                                      86      PRO CD   6.377       42.440                                                                               -23.636                                     87      SER CA   2.489       45.324                                                                               -25.529                                     87      SER D    0.162       45.513                                                                               -25.619                                     87      SER OG   3.591       45.143                                                                               -27.583                                     88      ALA CB   -0.163      43.510                                                                               -21.828                                     88      ALA C    -0.898      45.717                                                                               -22.690                                     89      SER N    -2.219      45.691                                                                               -22.678                                     89      SER CB   -4.343      46.903                                                                               -22.898                                     89      SER C    -3.136      46.780                                                                               -20.727                                     90      LEU N    -2.446      47.656                                                                               -20.037                                     90      LEU C    -3.483      48.430                                                                               -17.864                                     90      LEU CB   -0.951      48.273                                                                               -18.426                                     90      LEU CD1  -0.028      46.341                                                                               -17.219                                     91      TYR N    -4.264      47.944                                                                               -16.938                                     91      TYR C    -4.873      45.750                                                                               -14.685                                     91      TYR D    -4.496      47.749                                                                               -14.02                                      91      TYR CG   -7.094      48.237                                                                               -17.741                                     91      TYR CD2  -7.971      49.275                                                                               -18.149                                     91      TYR CE2  -8.315      49.421                                                                               -19.492                                     91      TYR OH   -8.182      48.752                                                                               -21.764                                     92      ALA CA   -4.549      50.199                                                                               -12.707                                     92      ALA D    -6.723      50.898                                                                               -12.050                                     93      VAL N    -5.959      48.993                                                                               -11.129                                     93      VAL C    -6.708      49.014                                                                               -8.899                                      93      VAL CB   -7.957      47.555                                                                               -10.611                                     93      VAL CG2  -8.195      47.370                                                                               -12.072                                     94      LYS CA   -6.378      50.464                                                                               -6.999                                      94      LYS O    -8.458      50.480                                                                               -5.783                                      94      LYS CG   -5.394      52.320                                                                               -5.467                                      94      LYS CE   -4.399      54.208                                                                               -4.199                                      95      VAL N    -6.909      49.071                                                                               -5.026                                      95      VAL C    -6.919      48.499                                                                               -2.568                                      95      VAL CB   -8.104      47.030                                                                               -4.319                                      95      VAL CG2  -6.900      46.100                                                                               -4.332                                      96      LEU CA   -4.782      49.103                                                                               -1.486                                      96      LEU O    -3.942      51.121                                                                               -2.336                                      96      LEU CG   -3.593      46.799                                                                               -2.072                                      96      LEU CO2  -4.489      46.082                                                                               -1.045                                      97      GLY CA   -3.890      52.307                                                                               0.287                                       97      GLY O    -1.619      51.463                                                                               0.165                                       98      ALA CB   -0.428      55.478                                                                               1.510                                       98      ALA C    0.188       53.118                                                                               1.917                                       99      ASP N    -0.504      52.573                                                                               2.912                                       99      ASP OD1  -2.730      50.902                                                                               4.003                                       99      ASP CB   -0.648      51.603                                                                               5.175                                       99      ASP C    0.146       50.165                                                                               3.320                                       100     GLY N    -0.424      49.883                                                                               2.168                                       100     GLY C    -1.520      47.651                                                                               2.002                                       101     SER N    -2.342      48.128                                                                               2.908                                       101     SER C    -4.759      47.894                                                                               2.532                                       101     SER CB   -3.716      47.447                                                                               4.817                                       102     GLY N    -5.821      47.092                                                                               2.577                                       102     GLY C    -8.166      46.536                                                                               2.528                                       103     GLN N    -9.377      47.058                                                                               2.498                                       103     GLN C    -10.963     45.232                                                                               2.022                                       103     GLN CB   -11.671     47.307                                                                               3.274                                       103     GLN CD   -12.360     49.104                                                                               4.915                                       103     GLN NE2  -13.419     49.197                                                                               4.112                                       104     TYR CA   -12.068     43.126                                                                               1.508                                       104     TYR D    -12.939     43.276                                                                               -0.687                                      104     TYR CG   -11.629     40.829                                                                               2.472                                       104     TYR CD2  -10.379     40.959                                                                               1.860                                       104     TYR CE2  -9.352      40.057                                                                               2.171                                       104     TYR OH   -8.481      38.191                                                                               3.324                                       105     SER CA   -14.877     45.166                                                                               -0.034                                      105     SER O    -14.759     45.935                                                                               -2.258                                      105     SER OG   -15.209     47.039                                                                               1.450                                       106     TRP CA   -12.421     47.391                                                                               -1.948                                      106     TRP D    -12.021     46.648                                                                               -4.245                                      106     TRP CG   -11.645     49.111                                                                               -0.206                                      106     TRP CD2  -10.658     49.812                                                                               0.581                                       106     TRP CE2  -11.359     50.573                                                                               1.561                                       106     TRP CZ2  -10.671     51.318                                                                               2.500                                       106     TRP CH2  -9.293      51.291                                                                               2.455                                       107     ILE CA   -10.765     44.250                                                                               -3.325                                      107     ILE O    -11.695     43.474                                                                               -5.398                                      107     ILE CG1  -8.634      43.784                                                                               -1.936                                      107     ILE CD1  -8.233      42.998                                                                               -0.627                                      91      TYR CB   -6.686      48.093                                                                               -16.314                                     91      TYR CD1  -6.595      47.415                                                                               -18.755                                     91      TYR CE1  -6.905      47.572                                                                               -20.090                                     91      TYR CZ   -7.794      48.582                                                                               -20.463                                     92      ALA N    -4.895      49.958                                                                               -14.104                                     92      ALA C    -5.823      50.033                                                                               -11.903                                     92      ALA CB   -3.997      51.621                                                                               -12.488                                     93      VAL CA   -7.183      48.854                                                                               -10.325                                     93      VAL D    -6.181      47.993                                                                               -8.372                                      93      VAL CG1  -9.213      47.488                                                                               -9.725                                      94      LYS N    -6.907      50.217                                                                               -8.327                                      94      LYS C    -7.331      49.985                                                                               -5.894                                      94      LYS CB   -6.051      51.976                                                                               -6.818                                      94      LYS CD   -4.868      53.785                                                                               -5.582                                      94      LYS NZ   -3.735      55.544                                                                               -4.387                                      95      VAL CA   -7.646      48.457                                                                               -3.920                                      95      VAL O    -7.425      48.156                                                                               -1.501                                      95      VAL CG1  -8.868      46.852                                                                               -5.619                                      96      LEU N    -5.676      48.974                                                                               -2.604                                      96      LEU C    -4.331      50.559                                                                               -1.321                                      96      LEU CB   -3.509      48.241                                                                               -1.573                                      96      LEU CD1  -2.207      46.184                                                                               -2.163                                      97      GLY N    -4.326      50.975                                                                               -0.086                                      97      GLY C    -2.363      52.437                                                                               0.385                                       98      ALA N    -1.954      53.648                                                                               0.758                                       98      ALA CA   -0.563      54.068                                                                               0.965                                       98      ALA O    1.393       52.921                                                                               1.663                                       99      ASP OD2  -2.631      51.042                                                                               6.151                                       99      ASP CG   -2.083      51.131                                                                               5.040                                       99      ASP CA   0.101       51.610                                                                               3.855                                       99      ASP O    0.735       49.313                                                                               4.029                                       100     GLY CA   -0.343      48.521                                                                               1.615                                       100     GLY O    -1.649      46.512                                                                               1.479                                       101     SER CA   -3.542      47.388                                                                               3.315                                       101     SER O    -4.758      48.972                                                                               1.907                                       101     SER OG   -4.411      48.634                                                                               5.209                                       102     GLY CA   -7.077      47.422                                                                               1.896                                       102     GLY O    -7.888      45.431                                                                               3.030                                       103     GLN CA   -10.535     46.297                                                                               3.020                                       103     GLN O    -10.779     45.482                                                                               0.817                                       103     GLN CG   -11.368     48.005                                                                               4.586                                       103     GLN OE1  -12.159     49.816                                                                               5.902                                       104     TYR N    -11.611     44.141                                                                               2.451                                       104     TYR C    -13.031     43.690                                                                               0.473                                       104     TYR CB   -12.697     41.866                                                                               2.143                                       104     TYR CD1  -11.819     39.789                                                                               3.377                                       104     TYR CE1  -10.805     38.885                                                                               3.707                                       104     TYR CZ   -9.564      39.022                                                                               3.081                                       105     SER N    -13.909     44.572                                                                               0.903                                       105     SER C    -14.172     45.920                                                                               -1.159                                      105     SER CB   -15.880     46.121                                                                               0.601                                       106     TRP N    -13.079     46.625                                                                               -0.834                                      106     TRP C    -11.895     46.436                                                                               -3.012                                      106     TRP CB   -11.321     48.254                                                                               -1.355                                      106     TRP CD1  -12.862     49.524                                                                               0.264                                       106     TRP NE1  -12.691     50.358                                                                               1.360                                       106     TRP CE3  -9.275      49.852                                                                               0.576                                       106     TRP CZ3  -8.568      50.563                                                                               1.525                                       107     ILE N    -11.339     45.330                                                                               -2.481                                      107     ILE C    -11.855     43.594                                                                               -4.190                                      107     ILE CB   -9.944      43.183                                                                               -2.523                                      107     ILE CG2  -9.632      41.930                                                                               -3.381                                      108     ILE N    -12.994     43.292                                                                               -3.577                                      108     ILE CA   -14.116     42.722                                                                               -4.32                                       108     ILE D    -14.894     43.329                                                                               -6.552                                      108     ILE CG1  -14.726     41.077                                                                               -2.482                                      108     ILE CD1  -15.452     40.845                                                                               -1.131                                      109     ASN CA   -15.204     46.018                                                                               -5.916                                      109     ASN O    -14.660     46.272                                                                               -8.235                                      109     ASN CG   -16.528     47.486                                                                               -4.353                                      109     ASN NO2  -16.633     48.447                                                                               -3.442                                      110     GLY CA   -11.952     45.917                                                                               -7.865                                      110     GLY D    -11.929     44.929                                                                               -10.034                                     111     ILE CA   -12.603     42.334                                                                               -9.099                                      111     ILE D    -13.921     42.384                                                                               -11.148                                     111     ILE CG1  -11.421     40.501                                                                               -7.655                                      111     ILE CD1  -11.588     39.706                                                                               -6.336                                      112     GLU CA   -16.118     43.376                                                                               -10.046                                     112     GLU D    -16.467     44.130                                                                               -12.246                                     112     GLU CG   -17.847     42.917                                                                               -8.135                                      112     GLU OE1  -19.041     40.866                                                                               -8.016                                      113     TRP N    -15.094     45.403                                                                               -10.971                                     113     TRP C    -14.076     45.663                                                                               -13.140                                     113     TRP CB   -13.882     47.553                                                                               -11.434                                     113     TRP CD1  -14.148     49.736                                                                               -12.681                                     113     TRP NE1  -13.597     50.443                                                                               -13.723                                     113     TRP CE3  -11.451     47.645                                                                               -13.809                                     113     TRP CZ3  -10.610     47.899                                                                               -14.879                                     114     ALA N    -13.089     44.801                                                                               -12.832                                     114     ALA C    -13.199     43.179                                                                               -14.752                                     114     ALA CB   -11.299     43.192                                                                               -13.140                                     115     ILE CA   -15.070     41.640                                                                               -14.897                                     115     ILE D    -16.077     42.225                                                                               -17.070                                     115     ILE CG1  -15.218     39.836                                                                               -13.043                                     115     ILE CD1  -16.004     39.411                                                                               -11.743                                     116     ALA CA   -17.390     44.440                                                                               -16.050                                     116     ALA D    -17.323     45.255                                                                               -18.343                                     117     ASN N    -15.423     45.390                                                                               -17.122                                     117     ASN C    -13.827     44.974                                                                               -19.034                                     117     ASN CB   -13.615     46.958                                                                               -17.426                                     117     ASN OD1  -14.565     49.082                                                                               -17.773                                     118     ASN N    -14.223     43.725                                                                               -18.967                                     118     ASN C    -12.240     42.444                                                                               -19.843                                     118     ASN CB   -14.247     42.863                                                                               -21.279                                     118     ASN OD1  -16.510     42.321                                                                               -20.759                                     119     MET N    -11.686     42.500                                                                               -18.675                                     119     MET C    -10.025     40.734                                                                               -18.928                                     119     MET CB   -9.810      42.461                                                                               -17.055                                     119     MET SD   -8.788      44.943                                                                               -17.526                                     120     ASP N    -8.904      40.437                                                                               -19.584                                     120     ASP C    -7.822      38.390                                                                               -18.856                                     120     ASP CB   -7.555      39.156                                                                               -21.236                                     120     ASP OD1  -7.801      40.706                                                                               -23.084                                     121     VAL N    -7.021      39.117                                                                               -18.115                                     121     VAL C    -6.296      39.534                                                                               -15.786                                     121     VAL CB   -4.755      38.587                                                                               -17.496                                     121     VAL CG2  -4.707      37.916                                                                               -18.846                                     122     ILE CA   -6.248      39.799                                                                               -13.397                                     122     ILE O    -4.829      38.012                                                                               -12.469                                     122     ILE CG1  -8.686      40.392                                                                               -13.063                                     122     ILE CD1  -9.976      39.788                                                                               -12.383                                     123     ASN CA   -3.145      39.854                                                                               -11.232                                     123     ASN O    -3.708      41.631                                                                               -9.833                                      123     ASN CG   -0.692      40.048                                                                               -10.777                                     123     ASN ND2  -0.346      40.747                                                                               -9.720                                      124     MET CA   -3.650      39.973                                                                               -7.438                                      108     ILE C    -14.639     43.694                                                                               -5.386                                      108     ILE CB   -15.246     42.265                                                                               -3.320                                      108     ILE CG2  -16.568     42.024                                                                               -4.095                                      109     ASN N    -14.751     44.958                                                                               -4.981                                      109     ASN C    -14.232     46.067                                                                               -7.084                                      109     ASN CB   -15.280     47.359                                                                               -5.207                                      109     ASN OD1  -17.455     46.695                                                                               -4.646                                      110     GLY N    -12.951     45.908                                                                               -6.774                                      110     GLY C    -12.108     44.712                                                                               -8.812                                      111     ILE N    -12.379     43.539                                                                               -8.246                                      111     ILE C    -13.859     42.560                                                                               -9.942                                      111     ILE CB   -12.734     40.948                                                                               -8.364                                      111     ILE CG2  -13.122     39.791                                                                               -9.347                                      112     GLU N    -14.893     43.075                                                                               -9.280                                      112     GLU C    -15.872     44.347                                                                               -11.171                                     112     GLU CB   -17.229     43.899                                                                               -9.141                                      112     GLU CD   -18.724     41.824                                                                               -8.685                                      112     GLU DE2  -19.123     41.928                                                                               -9.866                                      113     TRP CA   -14.756     46.400                                                                               -12.000                                     113     TRP O    -14.319     45.932                                                                               -14.332                                     113     TRP CG   -13.486     48.556                                                                               -12.481                                     113     TRP CD2  -12.441     49.552                                                                               -13.463                                     113     TRP CE2  -12.545     49.761                                                                               -14.215                                     113     TRP CZ2  -11.696     50.045                                                                               -15.274                                     113     TRP CH2  -10.752     49.074                                                                               -15.603                                     114     ALA CA   -12.333     44.065                                                                               -13.874                                     114     ALA O    -12.963     43.074                                                                               -15.978                                     115     ILE N    -14.174     42.540                                                                               -14.119                                     115     ILE C    -15.928     42.485                                                                               -15.856                                     115     ILE CB   -16.000     40.840                                                                               -13.922                                     115     ILE CG2  -17.151     40.168                                                                               -14.755                                     116     ALA N    -16.534     43.527                                                                               -15.267                                     116     ALA C    -16.706     45.069                                                                               -17.278                                     116     ALA CB   -18.011     45.510                                                                               -15.151                                     117     ASN CA   -14.553     45.967                                                                               -18.139                                     117     ASN O    -12.997     45.436                                                                               -19.820                                     117     ASN CG   -14.400     48.177                                                                               -16.939                                     117     ASN ND2  -14.931     45.249                                                                               -15.736                                     118     ASN CA   -13.760     42.642                                                                               -19.832                                     118     ASN O    -11.617     42.309                                                                               -20.932                                     118     ASN CG   -15.737     43.060                                                                               -21.395                                     118     ASN ND2  -16.136     44.096                                                                               -22.133                                     119     MET CA   -10.232     42.222                                                                               -18.478                                     119     MET O    -10.888     39.838                                                                               -18.759                                     119     MET CG   -9.880      43.883                                                                               -16.502                                     119     MET CB   -9.982      46.061                                                                               -18.263                                     120     ASP CA   -8.480      39.118                                                                               -20.030                                     120     ASP O    -8.038      37.189                                                                               -18.690                                     120     ASP CG   -8.237      39.730                                                                               -22.454                                     120     ASP OD2  -9.327      39.135                                                                               -22.739                                     121     VAL CA   -6.226      38.601                                                                               -16.974                                     121     VAL O    -6.284      40.788                                                                               -15.909                                     121     VAL CG1  -3.758      38.176                                                                               -16.427                                     122     ILE N    -6.318      38.978                                                                               -14.590                                     122     ILE C    -5.020      39.262                                                                               -12.627                                     122     ILE CB   -7.476      39.604                                                                               -12.466                                     122     ILE CG2  -7.221      39.883                                                                               -10.954                                     123     ASN N    -4.263      40.222                                                                               -12.110                                     123     ASN C    -3.502      40.404                                                                               -9.861                                      123     ASN CB   -1.828      40.478                                                                               -11.697                                     123     ASN OD1  -0.063      38.990                                                                               -11.018                                     124     MET N    -3.458      39.604                                                                               -8.832                                      124     MET C    -2.423      39.603                                                                               -6.614                                      124     MET O    -2.306      38.508                                                                               -6.09                                       124     MET CG   -6.158      40.082                                                                               -7.47                                       124     MET CE   -7.940      38.095                                                                               -7.542                                      125     SER CA   -0.193      40.287                                                                               -5.769                                      125     SER O    0.235       41.617                                                                               -3.805                                      125     SER DG   1.444       40.496                                                                               -7.575                                      126     LEU CA   -1.842      40.347                                                                               -2.386                                      126     LEU O    -2.844      38.136                                                                               -2.529                                      126     LEU CG   -3.988      41.447                                                                               -3.333                                      126     LEU CD2  -4.179      42.760                                                                               -4.073                                      127     GLY CA   -3.035      37.871                                                                               0.193                                       127     GLY O    -2.446      39.030                                                                               2.220                                       128     GLY CA   -4.475      37.496                                                                               3.642                                       128     GLY D    -4.903      35.158                                                                               3.276                                       129     PRO CA   -4.671      34.525                                                                               5.998                                       129     PRO D    -6.338      32.887                                                                               6.305                                       129     PRO CG   -4.419      36.116                                                                               7.727                                       130     SER N    -7.051      35.015                                                                               5.912                                       130     SER C    -9.218      34.884                                                                               4.726                                       130     SER CB   -9.069      35.351                                                                               7.216                                       131     GLY N    -10.083     33.967                                                                               4.349                                       131     GLY C    -12.205     34.713                                                                               3.542                                       132     SER N    -13.040     35.058                                                                               2.594                                       132     SER C    -15.289     34.805                                                                               1.936                                       132     SER CB   -14.590     36.927                                                                               3.145                                       133     ALA N    -16.547     34.588                                                                               2.294                                       133     ALA C    -17.650     34.965                                                                               0.097                                       133     ALA CB   -18.866     33.828                                                                               1.996                                       134     ALA CA   -17.872     37.259                                                                               -0.792                                      134     ALA D    -16.781     37.585                                                                               -2.869                                      135     LEU N    -15.478     37.229                                                                               -1.046                                      135     LEU C    -14.158     36.005                                                                               -2.705                                      135     LEU CB   -13.038     37.328                                                                               -0.798                                      135     LEU CD1  -11.460     38.415                                                                               -2.292                                      136     LYS N    -14.509     34.825                                                                               -2.173                                      136     LYS C    -15.544     33.739                                                                               -4.150                                      136     LYS CB   -14.903     32.341                                                                               -2.186                                      136     LYS CD   -15.083     29.892                                                                               -2.134                                      136     LYS NZ   -15.308     28.411                                                                               -4.160                                      137     ALA CA   -17.795     34.416                                                                               -4.883                                      137     ALA O    -17.705     35.049                                                                               -7.208                                      138     ALA N    -16.529     36.301                                                                               -5.729                                      138     ALA C    -14.903     36.696                                                                               -7.557                                      138     ALA CB   -15.522     38.567                                                                               -5.934                                      139     VAL CA   -12.946     35.291                                                                               -7.837                                      139     VAL D    -13.208     34.070                                                                               -9.877                                      139     VAL CG1  -10.919     33.856                                                                               -7.866                                      140     ASP N    -14.593     33.536                                                                               -8.122                                      140     ASP C    -16.023     33.131                                                                               -10.084                                     140     ASP CB   -16.149     31.549                                                                               -8.138                                      140     ASP OD1  -14.178     30.403                                                                               -7.202                                      141     LYS N    -16.658     34.263                                                                               -9.820                                      141     LYS C    -16.373     35.415                                                                               -11.946                                     141     LYS CB   -18.039     36.275                                                                               -10.325                                     141     LYS CD   -19.586     38.187                                                                               -10.536                                     141     LYS NZ   -21.138     40.037                                                                               -10.275                                     142     ALA CA   -14.173     36.192                                                                               -12.614                                     142     ALA D    -13.770     35.169                                                                               -14.755                                     143     VAL N    -13.582     33.886                                                                               -12.832                                     143     VAL C    -14.346     32.233                                                                               -14.496                                     143     VAL CB   -12.551     31.673                                                                               -12.714                                     143     VAL CG2  -11.305     32.195                                                                               -12.014                                     144     ALA CA   -16.744     31.834                                                                               -14.641                                     124     MET CB   -4.943      39.387                                                                               -6.890                                      124     MET SD   -7.585      39.472                                                                               -6.450                                      125     SER N    -1.454      40.496                                                                               -6.502                                      125     SER C    -0.422      40.712                                                                               -4.326                                      125     SER CB   1.021       41.027                                                                               -6.328                                      126     LEU N    -1.433      40.075                                                                               -3.775                                      126     LEU C    -2.438      39.056                                                                               -1.807                                      126     LEU CB   -2.791      41.568                                                                               -2.410                                      126     LEU CD1  -5.278      41.131                                                                               -2.578                                      127     GLY N    -2.522      39.082                                                                               -0.481                                      127     GLY C    -3.176      38.180                                                                               1.682                                       128     GLY N    -4.121      37.443                                                                               2.222                                       128     GLY C    -4.644      36.038                                                                               4.104                                       129     PRO N    -4.519      35.857                                                                               5.402                                       129     PRO C    -6.116      34.086                                                                               6.082                                       129     PRO CB   -4.060      34.684                                                                               7.384                                       129     PRO CD   -4.239      36.870                                                                               6.418                                       130     SER CA   -8.470      34.611                                                                               6.023                                       130     SER O    -8.949      35.881                                                                               4.029                                       130     SER OG   -8.723      34.626                                                                               8.403                                       131     GLY CA   -10.824     34.229                                                                               3.074                                       131     GLY O    -12.495     34.722                                                                               4.751                                       132     SER CA   -14.407     35.433                                                                               3.011                                       132     SER O    -14.799     34.586                                                                               0.824                                       132     SER OG   -14.693     37.539                                                                               1.875                                       133     ALA CA   -17.507     34.057                                                                               1.324                                       133     ALA O    -17.743     34.437                                                                               -1.014                                      134     ALA N    -17.683     36.288                                                                               0.294                                       134     ALA C    -16.635     37.369                                                                               -1.674                                      134     ALA CB   -18.263     38.600                                                                               -0.187                                      135     LEU CA   -14.197     37.244                                                                               -1.804                                      135     LEU O    -13.794     36.020                                                                               -3.890                                      135     LEU CG   -11.693     37.130                                                                               -1.508                                      135     LEU CD2  -10.582     36.807                                                                               -0.519                                      136     LYS CA   -14.543     33.597                                                                               -3.013                                      136     LYS O    -15.279     33.431                                                                               -5.305                                      136     LYS CG   -14.743     31.067                                                                               -3.043                                      136     LYS CE   -15.743     28.707                                                                               -2.778                                      137     ALA N    -16.744     34.260                                                                               -3.847                                      137     ALA C    -17.338     35.303                                                                               -6.045                                      137     ALA CB   -19.094     34.941                                                                               -4.263                                      138     ALA CA   -16.001     37.311                                                                               -6.685                                      138     ALA D    -14.985     36.843                                                                               -8.762                                      139     VAL N    -13.950     35.959                                                                               -7.027                                      139     VAL C    -13.623     34.228                                                                               -8.720                                      139     VAL CB   -11.830     34.671                                                                               -6.968                                      139     VAL CG2  -11.078     35.780                                                                               -6.253                                      140     ASP CA   -15.274     32.496                                                                               -8.929                                      140     ASP O    -16.080     32.579                                                                               -11.190                                     140     ASP CG   -15.388     30.640                                                                               -7.186                                      140     ASP OD2  -16.139     30.132                                                                               -6.329                                      141     LYS CA   -17.373     35.006                                                                               -10.868                                     141     LYS D    -16.700     35.248                                                                               -13.111                                     141     LYS CG   -18.884     37.056                                                                               -11.306                                     141     LYS CE   -20.572     39.051                                                                               -11.250                                     142     ALA N    -15.167     35.848                                                                               -11.566                                     142     ALA C    -13.818     35.010                                                                               -13.521                                     142     ALA CB   -12.870     36.697                                                                               -11.948                                     143     VAL CA   -13.168     32.705                                                                               -13.650                                     143     VAL O    -14.140     31.886                                                                               -15.639                                     143     VAL CG1  -12.300     30.370                                                                               -13.461                                     144     ALA N    -15.531     32.238                                                                               -13.875                                     144     ALA C    -16.928     32.681                                                                               -15.861                                     144     ALA D    -17.380     32.263                                                                               -16.958                                     145     SER N    -16.507     33.948                                                                               -15.706                                     145     SER C    -15.609     34.773                                                                               -17.829                                     145     SER CB   -17.016     36.376                                                                               -16.414                                     146     GLY N    -14.577     33.986                                                                               -17.565                                     146     GLY C    -12.273     34.491                                                                               -18.385                                     147     VAL N    -12.150     35.162                                                                               -17.254                                     147     VAL C    -9.850      34.836                                                                               -16.323                                     147     VAL CB   -11.152     36.977                                                                               -15.889                                     147     VAL CG2  -12.340     37.915                                                                               -16.230                                     148     VAL CA   -7.482      34.230                                                                               -16.008                                     148     VAL O    -6.840      36.133                                                                               -14.750                                     148     VAL CG1  -5.079      33.403                                                                               -16.281                                     149     VAL N    -7.258      34.355                                                                               -13.531                                     149     VAL C    -5.700      34.385                                                                               -11.613                                     149     VAL CB   -8.224      34.890                                                                               -11.315                                     149     VAL CG2  -9.456      35.386                                                                               -12.096                                     150     VAL CA   -3.393      34.987                                                                               -10.901                                     150     VAL O    -3.592      36.778                                                                               -9.400                                      150     VAL CG1  -0.973      34.633                                                                               -11.461                                     151     ALA N    -2.568      34.946                                                                               -8.595                                      151     ALA C    -1.080      35.036                                                                               -6.657                                      151     ALA CB   -3.557      35.390                                                                               -6.307                                      152     ALA CA   0.714       35.438                                                                               -5.112                                      152     ALA O    -0.728      34.466                                                                               -3.467                                      153     ALA N    1.125       33.302                                                                               -3.912                                      153     ALA C    0.931       32.725                                                                               -1.511                                      153     ALA CB   1.750       31.038                                                                               -3.195                                      154     GLY CA   2.043       34.211                                                                               0.125                                       154     GLY D    4.189       33.267                                                                               -0.118                                      155     ASN CA   5.344       34.787                                                                               2.037                                       155     ASN D    6.101       34.829                                                                               4.295                                       155     ASN CG   5.890       36.702                                                                               0.500                                       155     ASN ND2  5.454       37.965                                                                               0.352                                       156     GLU CA   4.633       32.537                                                                               4.970                                       156     GLU D    5.374       30.637                                                                               6.222                                       156     GLU CG   2.491       32.442                                                                               6.368                                       156     GLU DE1  1.744       34.322                                                                               5.312                                       157     GLY N    6.389       31.057                                                                               4.227                                       157     GLY C    6.503       28.622                                                                               4.553                                       158     THR N    7.147       27.793                                                                               5.382                                       158     THR OG1  8.707       25.487                                                                               6.217                                       158     THR CA   6.552       26.487                                                                               5.702                                       158     THR O    6.479       27.335                                                                               7.977                                       159     SER DG   3.141       25.904                                                                               10.525                                      159     SER CA   4.835       25.210                                                                               8.855                                       159     SER D    3.339       23.281                                                                               9.030                                       160     GLY CA   5.434       21.504                                                                               8.895                                       160     GLY O    4.806       21.326                                                                               6.555                                       161     SER CA   2.654       19.777                                                                               7.054                                       161     SER O    0.696       20.347                                                                               5.869                                       161     SER DG   1.854       18.028                                                                               8.585                                       162     SER CA   0.167       22.725                                                                               7.113                                       162     SER O    1.533       23.840                                                                               5.394                                       162     SER OG   0.184       23.091                                                                               9.480                                       163     SER CA   -0.611      24.750                                                                               3.990                                       163     SER O    -1.078      26.548                                                                               5.504                                       163     SER OG   -1.992      25.718                                                                               2.331                                       164     THR CA   0.609       28.340                                                                               4.312                                       164     THR O    0.485       30.502                                                                               3.278                                       164     THR OG1  2.984       28.282                                                                               3.692                                       165     VAL N    -0.513      28.742                                                                               2.190                                       165     VAL C    -2.028      30.545                                                                               1.497                                       144     ALA CB   -17.942     31.968                                                                               -13.700                                     145     SEP CA   -16.682     34.917                                                                               -16.786                                     145     SEP O    -15.910     35.321                                                                               -18.893                                     145     SEP OG   -15.882     36.955                                                                               -15.849                                     146     GLY CA   -13.619     33.799                                                                               -18.675                                     146     GLY D    -11.420     34.386                                                                               -19.266                                     147     VAL CA   -10.874     35.856                                                                               -16.912                                     147     VAL D    -10.171     33.991                                                                               -15.486                                     147     VAL CG1  -9.896      37.803                                                                               -15.570                                     148     VAL N    -8.583      35.018                                                                               -16.603                                     148     VAL C    -7.157      34.907                                                                               -14.701                                     148     VAL CB   -6.273      34.126                                                                               -16.950                                     148     VAL CG2  -6.590      33.432                                                                               -18.262                                     149     VAL CA   -6.987      34.965                                                                               -12.249                                     149     VAL D    -5.624      33.173                                                                               -11.439                                     149     VAL CG1  -7.893      35.619                                                                               -10.009                                     150     VAL N    -4.732      35.301                                                                               -11.404                                     150     VAL C    -3.157      35.625                                                                               -9.559                                      150     VAL CB   -2.274      35.305                                                                               -11.951                                     150     VAL CG2  -2.675      34.843                                                                               -13.301                                     151     ALA CA   -2.361      35.582                                                                               -7.287                                      151     ALA D    -0.618      33.889                                                                               -6.904                                      152     ALA N    -0.490      35.907                                                                               -5.822                                      152     ALA C    0.304       34.320                                                                               -4.158                                      152     ALA CB   1.266       36.607                                                                               -4.294                                      153     ALA CA   0.840       32.250                                                                               -2.943                                      153     ALA O    0.317       32.192                                                                               -0.599                                      154     GLY N    1.827       33.693                                                                               -1.244                                      154     GLY C    3.519       34.069                                                                               0.550                                       155     ASN N    3.958       34.788                                                                               1.568                                       155     ASN C    5.399       34.258                                                                               3.462                                       155     ASN CB   6.008       36.158                                                                               1.904                                       155     ASN OD1  6.123       36.065                                                                               -0.534                                      156     GLU N    4.711       33.168                                                                               3.675                                       156     GLU C    5.522       31.328                                                                               5.183                                       156     GLU CB   3.205       31.980                                                                               5.100                                       156     GLU CD   2.394       33.951                                                                               6.270                                       156     GLU DE2  3.106       34.456                                                                               7.146                                       157     GLY CA   7.306       29.917                                                                               4.387                                       157     GLY O    5.416       28.346                                                                               4.009                                       158     THR CG2  8.079       25.396                                                                               3.850                                       158     THR CB   7.564       25.346                                                                               5.296                                       158     THR C    6.100       26.480                                                                               7.157                                       159     SER N    5.338       25.441                                                                               7.497                                       159     SER CB   3.673       26.105                                                                               9.212                                       159     SER C    4.494       23.720                                                                               8.944                                       160     GLY N    5.574       22.967                                                                               8.835                                       160     GLY C    4.576       21.045                                                                               7.738                                       161     SER N    3.525       20.310                                                                               8.116                                       161     SER C    1.477       20.708                                                                               6.786                                       161     SER CB   2.344       18.293                                                                               7.271                                       162     SER N    1.303       21.841                                                                               7.459                                       162     SER C    0.430       23.552                                                                               5.848                                       162     SER CB   -0.213      23.666                                                                               8.242                                       163     SER N    -0.679      23.921                                                                               5.197                                       163     SER C    -0.441      26.177                                                                               4.513                                       163     SER CB   -1.890      24.642                                                                               3.211                                       164     THR N    0.387       26.952                                                                               3.852                                       164     THR C    0.185       29.286                                                                               3.194                                       164     THR CB   2.095       28.518                                                                               4.818                                       164     THR CG2  2.397       27.610                                                                               6.001                                       165     VAL CA   -0.959      29.542                                                                               1.010                                       165     VAL O    -2.929      30.132                                                                               2.280                                       165     VAL CB   -1.339      28.624                                                                               -0.161                                      165     VAL CG2  -0.210      27.716                                                                               -0.699                                      166     GLY CA   -2.945      32.778                                                                               1.626                                       166     GLY D    -4.124      32.106                                                                               -0.396                                      167     TYR CA   -6.223      34.046                                                                               0.113                                       167     TYR O    -5.474      36.283                                                                               0.084                                       167     TYR CG   -7.791      32.964                                                                               1.709                                       167     TYR CD2  -8.710      32.116                                                                               1.133                                       167     TYR CE2  -9.068      30.955                                                                               1.809                                       167     TYR DH   -8.880      29.481                                                                               3.658                                       168     PRO CG   -6.943      36.376                                                                               -3.938                                      168     PRO CB   -7.964      35.344                                                                               -3.505                                      168     PRO C    -6.398      33.336                                                                               -3.270                                      169     GLY N    -5.086      33.193                                                                               -3.189                                      169     GLY C    -4.937      30.702                                                                               -3.470                                      170     LYS N    -5.402      30.579                                                                               -2.255                                      170     LYS C    -7.055      28.773                                                                               -2.516                                      170     LYS CB   -6.246      29.294                                                                               -0.286                                      170     LYS CD   -6.250      28.289                                                                               2.031                                       170     LYS NZ   -4.259      27.463                                                                               3.215                                       171     TYR CA   -9.012      29.043                                                                               -3.859                                      171     TYR O    -7.760      28.714                                                                               -5.928                                      171     TYR CG   -10.497     30.984                                                                               -3.047                                      171     TYR CD2  -10.456     32.374                                                                               -3.026                                      171     TYR CE2  -10.941     33.088                                                                               -1.936                                      171     TYR OH   -12.008     33.119                                                                               0.170                                       172     PRO CA   -9.093      26.417                                                                               -6.596                                      172     PRO O    -8.525      26.784                                                                               -8.881                                      172     PRO CG   -10.600     25.271                                                                               -5.096                                      173     SER N    -10.097     28.167                                                                               -8.019                                      173     SER C    -9.025      29.773                                                                               -9.595                                      173     SER CB   -11.528     29.623                                                                               -9.481                                      174     VAL N    -8.162      29.944                                                                               -8.614                                      174     VAL C    -5.754      30.131                                                                               -9.068                                      174     VAL CB   -6.899      31.775                                                                               -7.596                                      174     VAL CG2  -8.220      32.503                                                                               -7.323                                      175     ILE CA   -3.569      30.156                                                                               -10.024                                     175     ILE D    -2.450      31.958                                                                               -8.955                                      175     ILE CG1  -3.857      29.978                                                                               -12.524                                     175     ILE CD1  -3.692      30.529                                                                               -13.946                                     176     ALA CA   -1.335      30.517                                                                               -6.870                                      176     ALA O    0.453       29.215                                                                               -7.838                                      177     VAL N    0.864       31.410                                                                               -7.180                                      177     VAL C    3.225       31.693                                                                               -6.473                                      177     VAL CB   2.439       32.607                                                                               -8.755                                      177     VAL CG2  1.374       32.552                                                                               -9.845                                      178     GLY CA   5.168       30.703                                                                               -5.339                                      178     GLY O    6.499       31.435                                                                               -7.286                                      179     ALA CA   8.715       32.037                                                                               -5.859                                      179     ALA C    10.198      30.481                                                                               -4.719                                      180     VAL N    10.659      31.162                                                                               -6.885                                      180     VAL C    13.048      31.585                                                                               -7.171                                      180     VAL CB   12.075      29.514                                                                               -8.166                                      180     VAL CG2  11.675      30.129                                                                               -9.500                                      181     ASP CA   15.451      32.108                                                                               -7.039                                      181     ASP O    15.339      31.090                                                                               -9.292                                      181     ASP CG   17.120      30.534                                                                               -5.971                                      181     ASP OD2  17.680      30.256                                                                               -4.887                                      182     SER CA   17.622      32.214                                                                               -10.191                                     182     SER O    18.365      30.452                                                                               -11.670                                     182     SER OG   18.016      34.561                                                                               -10.475                                     183     SER CA   18.716      28.645                                                                               -9.444                                      183     SER O    17.859      26.415                                                                               -9.397                                      165     VAL CG1  -1.947      29.357                                                                               -1.374                                      166     GLY N    -1.910      31.821                                                                               1.129                                       166     GLY C    -4.098      32.859                                                                               0.617                                       167     TYR N    -5.054      33.730                                                                               0.970                                       167     TYR C    -5.993      35.389                                                                               -0.606                                      167     TYR CB   -7.464      34.252                                                                               0.964                                       167     TYR CD1  -7.208      32.703                                                                               2.947                                       167     TYR CE1  -7.567      31.528                                                                               3.615                                       167     TYR CZ   -8.486      30.671                                                                               3.046                                       168     PRO N    -6.380      35.499                                                                               -1.850                                      168     PRO CD   -6.273      36.752                                                                               -2.624                                      168     PRO CA   -7.134      34.457                                                                               -2.560                                      168     PRO D    -7.097      32.520                                                                               -3.912                                      169     GLY CA   -4.446      32.077                                                                               -3.927                                      169     GLY O    -4.880      29.733                                                                               -4.249                                      170     LYS CA   -5.856      29.265                                                                               -1.745                                      170     LYS D    -7.308      27.554                                                                               -2.524                                      170     LYS CG   -5.795      28.106                                                                               0.585                                       170     LYS CE   -5.731      27.271                                                                               3.029                                       171     TYR N    -7.838      29.616                                                                               -3.148                                      171     TYR C    -8.603      28.309                                                                               -5.113                                      171     TYR CB   -9.962      30.224                                                                               -4.242                                      171     TYR CD1  -11.060     30.303                                                                               -1.982                                      171     TYR CE1  -11.520     31.003                                                                               -0.867                                      171     TYR CZ   -11.528     32.398                                                                               -0.886                                      172     PRO N    -9.297      27.204                                                                               -5.374                                      172     PRO C    -9.233      27.156                                                                               -7.909                                      172     PRO CB   -10.167     25.329                                                                               -6.513                                      172     PRO CD   -10.364     26.669                                                                               -4.514                                      173     SER CA   -10.220     28.818                                                                               -9.330                                      173     SER O    -8.966      30.233                                                                               -10.742                                     173     SER OG   -11.595     30.546                                                                               -8.406                                      174     VAL CA   -7.053      30.891                                                                               -8.855                                      174     VAL O    -5.612      29.152                                                                               -8.344                                      174     VAL CG1  -5.796      32.837                                                                               -7.617                                      175     ILE N    -4.911      30.729                                                                               -9.885                                      175     ILE C    -2.714      30.736                                                                               -8.894                                      175     ILE CB   -2.953      30.524                                                                               -11.419                                     175     ILE CG2  -1.451      30.089                                                                               -11.512                                     176     ALA N    -2.220      30.028                                                                               -7.925                                      176     ALA C    0.120       30.301                                                                               -7.310                                      176     ALA CB   -1.639      29.838                                                                               -5.541                                      177     VAL CA   2.261       31.534                                                                               -7.656                                      177     VAL D    3.178       32.657                                                                               -5.721                                      177     VAL CG1  3.842       32.667                                                                               -9.392                                      178     GLY N    4.077       30.654                                                                               -6.358                                      178     GLY C    6.446       31.233                                                                               -6.074                                      179     ALA N    7.512       31.447                                                                               -5.287                                      179     ALA C    9.939       31.099                                                                               -5.779                                      179     ALA CB   9.025       33.251                                                                               -4.973                                      180     VAL CA   11.970      30.482                                                                               -6.981                                      180     VAL D    12.712      32.691                                                                               -7.627                                      180     VAL CG1  11.271      28.251                                                                               -7.855                                      181     ASP N    14.267      31.203                                                                               -6.800                                      181     ASP C    15.942      31.804                                                                               -8.462                                      181     ASP CB   16.446      31.921                                                                               -5.914                                      181     ASP OD1  17.105      29.785                                                                               -6.972                                      182     SER N    17.087      32.386                                                                               -8.847                                      182     SER C    18.153      30.817                                                                               -10.494                                     182     SER CB   18.678      33.313                                                                               -10.464                                     183     SER N    18.258      30.042                                                                               -9.423                                      183     SER C    17.581      27.614                                                                               -9.547                                      183     SER CB   19.256      28.323                                                                               -8.007                                      183     SER OG   20.589      28.615                                                                               -8.251                                      184     ASN CA   15.144      27.317                                                                               -9.580                                      184     ASN D    14.138      25.759                                                                               -8.097                                      184     ASN CG   14.990      26.998                                                                               -12.076                                     184     ASN ND2  15.352      26.210                                                                               -13.076                                     185     GLN CA   15.276      26.646                                                                               -5.835                                      185     GLN O    14.159      28.726                                                                               -5.386                                      185     GLN CG   16.539      26.242                                                                               -3.614                                      185     GLN OE1  18.864      25.799                                                                               -4.061                                      186     ARG N    13.278      26.958                                                                               -4.448                                      186     ARG C    12.780      25.782                                                                               -2.866                                      186     ARG CB   11.215      26.843                                                                               -3.116                                      186     ARG CD   9.467       26.337                                                                               -1.468                                      186     ARG CZ   9.961       26.879                                                                               1.059                                       186     ARG NH2  10.966      26.321                                                                               1.783                                       187     ALA CA   12.728      31.064                                                                               -1.895                                      187     ALA O    11.158      30.043                                                                               -0.387                                      188     SER N    13.051      30.770                                                                               0.549                                       188     SER C    11.356      30.847                                                                               2.412                                       188     SER CB   13.767      30.456                                                                               2.938                                       189     PHE N    10.943      32.010                                                                               1.974                                       189     PHE C    8.499       32.198                                                                               1.609                                       189     PHE CB   9.787       34.217                                                                               2.243                                       189     PHE CD1  9.147       34.830                                                                               -0.121                                      189     PHE CE1  9.483       35.187                                                                               -1.411                                      189     PHE CZ   10.786      35.586                                                                               -1.725                                      190     SER CA   7.626       31.096                                                                               -0.391                                      190     SER O    7.034       29.083                                                                               0.866                                       190     SER OG   7.136       30.337                                                                               -2.618                                      191     SER CA   4.341       29.686                                                                               0.987                                       191     SER O    4.543       28.268                                                                               -0.995                                      191     SER OG   2.729       31.285                                                                               1.954                                       192     VAL CA   3.629       25.932                                                                               0.391                                       192     VAL D    1.559       25.698                                                                               1.598                                       192     VAL CG1  6.144       25.727                                                                               0.722                                       193     GLY N    1.938       24.172                                                                               0.047                                       193     GLY C    0.081       23.029                                                                               -0.901                                      194     PRO N    -1.023      22.283                                                                               -0.722                                      194     PRO C    -2.237      22.605                                                                               -2.914                                      194     PRO CB   -2.769      20.783                                                                               -1.210                                      194     PRO CD   -1.633      21.954                                                                               0.578                                       195     GLU CA   -3.145      24.850                                                                               -3.252                                      195     GLU O    -2.516      26.398                                                                               -4.936                                      195     GLU CG   -4.942      25.134                                                                               -1.435                                      195     GLU OE1  -3.110      24.960                                                                               0.165                                       196     LEU N    -0.829      25.264                                                                               -3.870                                      196     LEU C    0.228       25.376                                                                               -6.059                                      196     LEU CB   1.540       25.739                                                                               -3.854                                      196     LEU CD1  2.739       27.716                                                                               -4.639                                      197     ASP N    0.140       26.208                                                                               -7.093                                      197     ASP C    1.307       25.738                                                                               -9.293                                      197     ASP CB   -1.067      26.598                                                                               -9.191                                      197     ASP OD1  -2.804      25.155                                                                               -8.354                                      198     VAL N    2.013       26.889                                                                               -9.344                                      198     VAL C    4.157       27.950                                                                               -9.514                                      198     VAL CB   2.834       27.476                                                                               -11.637                                     198     VAL CG2  2.337       28.919                                                                               -11.484                                     199     MET CA   6.439       28.802                                                                               -9.498                                      199     MET O    6.696       29.518                                                                               -11.793                                     199     MET CG   7.365       26.849                                                                               -8.139                                      199     MET CE   8.227       27.755                                                                               -5.587                                      200     ALA CA   7.991       31.929                                                                               -11.055                                     200     ALA O    9.127       32.524                                                                               -9.060                                      184     ASN N    16.373      28.094                                                                               -9.602                                      184     ASN C    14.931      26.720                                                                               -8.197                                      184     ASN CB   15.014      26.341                                                                               -10.722                                     184     ASN OD1  14.700      28.184                                                                               -12.277                                     185     GLN N    15.542      27.247                                                                               -7.159                                      185     GLN C    14.200      27.494                                                                               -5.203                                      185     GLN CB   16.599      26.568                                                                               -5.101                                      185     GLN CD   18.011      26.102                                                                               -3.206                                      185     GLN NE2  18.266      26.386                                                                               -1.934                                      186     ARG CA   12.185      27.774                                                                               -3.841                                      186     ARG O    13.698      28.384                                                                               -2.093                                      186     ARG CG   10.214      27.471                                                                               -2.161                                      186     ARG NE   9.866       26.333                                                                               -0.117                                      186     ARG NH1  9.367       27.880                                                                               1.658                                       187     ALA N    12.294      30.009                                                                               -2.853                                      187     ALA C    12.262      30.604                                                                               -0.517                                      187     ALA CB   12.144      32.402                                                                               -2.344                                      188     SER CA   12.671      30.286                                                                               1.868                                       188     SER O    10.740      30.111                                                                               3.212                                       188     SER OG   14.137      31.826                                                                               2.841                                       189     PHE CA   9.697       32.688                                                                               2.418                                       189     PHE O    7.389       32.556                                                                               2.011                                       189     PHE CG   10.117      34.696                                                                               0.867                                       189     PHE CD2  11.415      35.116                                                                               0.567                                       189     PHE CE2  11.769      35.545                                                                               -0.701                                      190     SER N    8.703       31.526                                                                               0.499                                       190     SER C    6.663       30.162                                                                               0.328                                       190     SER CB   8.181       30.590                                                                               -1.708                                      191     SER N    5.388       30.551                                                                               0.326                                       191     SER C    4.261       28.330                                                                               0.223                                       191     SER CB   3.015       30.411                                                                               0.911                                       192     VAL N    3.756       27.310                                                                               0.928                                       192     VAL C    2.254       25.291                                                                               0.686                                       192     VAL CB   4.781       25.127                                                                               1.088                                       192     VAL CG2  4.617       25.104                                                                               2.592                                       193     GLY CA   0.629       23.564                                                                               0.410                                       193     GLY D    0.530       23.244                                                                               -2.015                                      194     PRO CA   -1.662      21.651                                                                               -1.873                                      194     PRO O    -2.403      22.244                                                                               -4.085                                      194     PRO CG   -2.311      20.622                                                                               0.213                                       195     GLU N    -2.522      23.793                                                                               -2.439                                      195     GLU C    -2.095      25.631                                                                               -4.058                                      195     GLU CB   -4.043      25.786                                                                               -2.470                                      195     GLU CD   -4.315      24.860                                                                               -0.100                                      195     GLU OE2  -5.138      24.520                                                                               0.785                                       196     LEU CA   0.241       25.929                                                                               -4.664                                      196     LEU O    0.305       24.121                                                                               -6.153                                      196     LEU CG   2.770       26.178                                                                               -4.643                                      196     LEU CD2  4.027       25.721                                                                               -3.911                                      197     ASP CA   0.032       25.774                                                                               -8.480                                      197     ASP O    1.655       24.734                                                                               -9.914                                      197     ASP CG   -2.406      26.351                                                                               -8.549                                      197     ASP OD2  -3.035      27.327                                                                               -8.088                                      198     VAL CA   3.206       26.970                                                                               -10.209                                     198     VAL O    3.752       28.699                                                                               -8.587                                      198     VAL CG1  1.930       26.726                                                                               -12.537                                     199     MET N    5.374       27.916                                                                               -10.016                                     199     MET C    6.845       29.810                                                                               -10.578                                     199     MET CB   7.660       27.970                                                                               -9.077                                      199     MET SD   6.755       27.449                                                                               -6.568                                      200     ALA N    7.426       30.942                                                                               -10.103                                     200     ALA C    9.088       32.666                                                                               -10.272                                     200     ALA CB   6.932       32.870                                                                               -11.638                                     201     PRO N    9.927       33.455                                                                               -10.95                                      201     PRO C    10.450      35.127                                                                               -9.23                                       201     PRO CB   11.817      34.723                                                                               -11.400                                     201     PRO CD   9.941       33.616                                                                               -12.405                                     202     GLY CA   10.473      36.204                                                                               -7.044                                      202     GLY O    11.352      37.124                                                                               -4.979                                      203     VAL CA   13.948      36.929                                                                               -5.716                                      203     VAL C    15.133      37.731                                                                               -7.593                                      203     VAL CG1  16.096      36.106                                                                               -4.612                                      204     SER N    14.865      39.182                                                                               -5.859                                      204     SER C    15.067      40.619                                                                               -7.872                                      204     SER CB   17.087      39.976                                                                               -6.326                                      205     ILE N    13.771      40.965                                                                               -8.008                                      205     ILE C    13.207      42.749                                                                               -9.478                                      205     ILE CB   11.532      40.833                                                                               -9.144                                      205     ILE CG2  10.899      41.281                                                                               -10.467                                     206     GLN N    13.956      43.095                                                                               -10.489                                     206     GLN C    13.002      44.978                                                                               -11.630                                     206     GLN CB   15.455      44.708                                                                               -11.740                                     206     GLN CD   17.285      45.145                                                                               -10.007                                     206     GLN NE2  16.556      46.260                                                                               -9.857                                      207     SER CA   11.217      46.571                                                                               -11.987                                     207     SER O    11.919      48.657                                                                               -11.004                                     207     SER OG   8.993       46.056                                                                               -12.613                                     208     THR CG2  9.171       50.339                                                                               -14.754                                     208     THR CB   8.620       50.415                                                                               -13.357                                     208     THR C    9.197       50.488                                                                               -10.803                                     209     LEU N    9.656       51.613                                                                               -10.228                                     209     LEU C    8.673       53.610                                                                               -9.262                                      209     LEU CB   10.335      52.192                                                                               -7.958                                      209     LEU CD1  11.968      51.114                                                                               -6.472                                      210     PRO N    7.790       54.139                                                                               -8.444                                      210     PRO C    8.383       56.573                                                                               -8.639                                      210     PRO CB   6.302       55.733                                                                               -7.517                                      210     PRO CD   7.193       53.491                                                                               -7.271                                      211     GLY CA   9.069       58.765                                                                               -9.410                                      211     GLY O    11.176      59.005                                                                               -10.259                                     212     ASN CA   10.903      57.422                                                                               -12.643                                     212     ASN O    13.188      57.181                                                                               -12.420                                     212     ASN CG   11.803      58.185                                                                               -14.814                                     212     ASN ND2  12.273      59.159                                                                               -15.576                                     213     LYS CA   12.810      54.946                                                                               -10.537                                     213     LYS O    11.775      53.039                                                                               -11.613                                     213     LYS CG   13.206      56.694                                                                               -8.767                                      213     LYS CE   14.105      58.218                                                                               -6.870                                      214     TYR N    13.681      52.703                                                                               -10.444                                     214     TYR C    14.383      50.600                                                                               -9.489                                      214     TYR CB   14.641      50.981                                                                               -11.984                                     214     TYR CD1  14.689      52.847                                                                               -13.678                                     214     TYR CE1  14.230      53.475                                                                               -14.814                                     214     TYR CZ   13.204      52.895                                                                               -15.550                                     215     GLY N    14.058      49.347                                                                               -9.158                                      215     GLY C    14.130      47.325                                                                               -7.749                                      216     ALA N    14.810      46.638                                                                               -6.831                                      216     ALA C    13.682      44.922                                                                               -5.512                                      216     ALA CB   15.715      44.354                                                                               -6.887                                      217     TYR CA   11.964      43.488                                                                               -4.440                                      217     TYR O    12.202      41.442                                                                               -5.656                                      217     TYR CG   10.117      45.291                                                                               -4.214                                      217     TYR CD2  9.016       45.933                                                                               -4.785                                      217     TYR CE2  8.654       47.219                                                                               -4.381                                      217     TYR OH   8.953       49.160                                                                               -2.988                                      218     ASN CA   11.640      39.942                                                                               -3.227                                      201     PRO CA   11.013      34.130                                                                               -10.238                                     201     PRO D    9.579       35.907                                                                               -9.682                                      201     PRO CG   11.392      34.040                                                                               -12.678                                     202     GLY N    10.925      35.204                                                                               -8.021                                      202     GLY C    11.580      36.658                                                                               -6.115                                      203     VAL N    12.815      36.503                                                                               -6.613                                      203     VAL C    14.706      38.017                                                                               -6.469                                      203     VAL CB   14.814      35.688                                                                               -5.351                                      203     VAL CG2  14.079      34.741                                                                               -4.378                                      204     SER CA   15.572      40.281                                                                               -6.487                                      204     SER O    15.786      40.685                                                                               -8.889                                      204     SER OG   17.752      41.186                                                                               -6.672                                      205     ILE CA   13.069      41.234                                                                               -9.225                                      205     ILE O    12.675      43.498                                                                               -8.648                                      205     ILE CG1  11.436      39.336                                                                               -8.810                                      205     ILE CD1  12.257      38.412                                                                               -9.771                                      206     GLN CA   14.204      44.517                                                                               -10.834                                     206     GLN O    12.669      44.318                                                                               -12.621                                     206     GLN CG   16.684      44.163                                                                               -10.980                                     206     GLN OE1  18.328      44.936                                                                               -9.353                                      207     SER N    12.359      46.064                                                                               -11.214                                     207     SER C    11.089      48.093                                                                               -11.749                                     207     SER CB   9.918       45.853                                                                               -11.569                                     208     THR N    10.054      48.664                                                                               -12.326                                     208     THR OG1  7.570       49.414                                                                               -13.144                                     208     THR CA   9.675       50.092                                                                               -12.173                                     208     THR O    8.423       49.807                                                                               -10.049                                     209     LEU CA   9.192       52.158                                                                               -8.959                                      209     LEU D    9.140       54.227                                                                               -10.222                                     209     LEU CG   10.804      50.816                                                                               -7.416                                      209     LEU CD2  9.607       50.282                                                                               -6.649                                      210     PRO CA   7.273       55.517                                                                               -8.649                                      210     PRO O    9.491       56.445                                                                               -8.104                                      210     PRO CG   6.004       54.379                                                                               -6.944                                      211     GLY N    8.077       57.665                                                                               -9.355                                      211     GLY C    10.094      58.454                                                                               -10.490                                     212     ASN N    9.851       57.770                                                                               -11.587                                     212     ASN C    12.059      56.753                                                                               -12.056                                     212     ASN CB   11.224      58.595                                                                               -13.499                                     212     ASN OD1  11.853      57.054                                                                               -15.323                                     213     LYS N    11.803      55.749                                                                               -11.247                                     213     LYS C    12.668      53.459                                                                               -10.866                                     213     LYS CB   12.769      55.241                                                                               -9.059                                      213     LYS CD   13.246      57.030                                                                               -7.312                                      213     LYS NZ   15.048      58.705                                                                               -7.921                                      214     TYR CA   13.800      51.246                                                                               -10.722                                     214     TYR O    15.211      51.253                                                                               -8.817                                      214     TYR CG   14.130      51.621                                                                               -13.246                                     214     TYR CD2  13.129      51.065                                                                               -14.014                                     214     TYR CE2  12.654      51.669                                                                               -15.178                                     214     TYR OH   12.756      53.458                                                                               -16.696                                     215     GLY CA   14.622      48.772                                                                               -7.905                                      215     GLY O    13.249      46.917                                                                               -8.521                                      216     ALA CA   14.454      45.203                                                                               -6.781                                      216     ALA O    13.948      45.527                                                                               -4.475                                      217     TYR N    12.758      43.982                                                                               -5.575                                      217     TYR C    12.033      41.928                                                                               -4.547                                      217     TYR CB   10.473      43.862                                                                               -4.570                                      217     TYR CD1  10.846      45.991                                                                               -3.236                                      217     TYR CE1  10.459      47.267                                                                               -2.790                                      217     TYR CZ   9.358       47.882                                                                               -3.391                                      218     ASN N    11.750      41.386                                                                               -3.391                                      218     ASN C    10.204      39.636                                                                               -2.749                                      218     ASN O    9.763       40.347                                                                               -1.81                                       218     ASN CG   14.031      39.566                                                                               -2.34                                       218     ASN ND2  14.660      39.644                                                                               -1.165                                      219     GLY CA   8.382       38.130                                                                               -2.649                                      219     GLY O    7.873       37.500                                                                               -4.876                                      220     THR CA   5.697       35.936                                                                               -4.179                                      220     THR O    4.417       36.742                                                                               -5.958                                      220     THR OG1  4.136       35.543                                                                               -2.451                                      221     SER N    4.738       38.238                                                                               -4.303                                      221     SER C    4.760       39.641                                                                               -6.383                                      221     SER CB   3.323       40.383                                                                               -4.546                                      222     MET N    6.060       39.389                                                                               -6.485                                      222     MET SD   7.768       41.533                                                                               -4.993                                      222     MET CB   8.351       40.015                                                                               -7.218                                      222     MET C    6.877       38.435                                                                               -8.567                                      223     ALA N    6.554       37.246                                                                               -8.041                                      223     ALA C    5.200       36.068                                                                               -9.707                                      223     ALA CB   6.509       34.807                                                                               -7.923                                      224     SER CA   2.758       36.488                                                                               -9.700                                      224     SER O    2.145       36.593                                                                               -12.057                                     224     SER OG   0.492       36.899                                                                               -9.157                                      225     PRO CA   3.095       39.130                                                                               -12.439                                     225     PRO O    3.406       38.650                                                                               -14.804                                     225     PRO CG   4.411       40.402                                                                               -10.764                                     226     HIS N    4.769       37.626                                                                               -13.299                                     226     HIS C    4.418       35.947                                                                               -15.061                                     226     HIS CB   6.608       36.046                                                                               -13.765                                     226     HIS ND1  8.048       37.488                                                                               -12.170                                     226     HIS CE1  9.270       38.052                                                                               -12.236                                     227     VAL N    3.593       35.366                                                                               -14.199                                     227     VAL C    1.479       35.197                                                                               -15.421                                     227     VAL CB   2.103       33.444                                                                               -13.619                                     227     VAL CG2  3.204       32.665                                                                               -12.891                                     228     ALA CA   0.011       37.109                                                                               -15.517                                     228     ALA D    -0.253      37.435                                                                               -17.828                                     229     GLY N    1.791       38.028                                                                               -16.941                                     229     GLY C    2.420       37.197                                                                               -19.187                                     230     ALA N    2.711       35.988                                                                               -18.646                                     230     ALA C    1.424       34.500                                                                               -20.153                                     230     ALA CB   3.298       33.624                                                                               -18.709                                     231     ALA CA   -1.010      34.416                                                                               -19.744                                     231     ALA O    -1.909      35.056                                                                               -21.852                                     232     ALA N    -0.778      36.657                                                                               -20.721                                     232     ALA C    -0.281      37.284                                                                               -23.078                                     232     ALA CB   -0.742      39.121                                                                               -21.377                                     233     LEU CA   1.617       36.293                                                                               -24.209                                     233     LEU O    0.696       35.231                                                                               -26.111                                     233     LEU CG   3.996       36.994                                                                               -23.453                                     233     LEU CD2  4.241       37.853                                                                               -24.680                                     234     ILE CD1  0.306       30.664                                                                               -21.657                                     234     ILE CB   -0.811      32.014                                                                               -23.570                                     234     ILE CA   -0.406      33.076                                                                               -24.644                                     234     ILE O    -1.883      33.144                                                                               -26.544                                     235     LEU CA   -3.596      35.028                                                                               -25.423                                     235     LEU O    -4.109      35.914                                                                               -27.589                                     235     LEU CG   -5.140      34.899                                                                               -23.342                                     235     LEU CD2  -6.252      34.138                                                                               -24.120                                     236     SER CA   -1.764      37.237                                                                               -27.986                                     236     SER O    -1.746      36.634                                                                               -30.290                                     236     SER OG   0.599       37.571                                                                               -27.582                                     237     LYS CA   -0.846      34.085                                                                               -29.95                                      237     LYS O    -2.378      32.951                                                                               -31.44                                      237     LYS CG   0.677       32.240                                                                               -30.71                                      218     ASN CB   12.553      39.340                                                                               -2.154                                      218     ASN OD1  14.612      39.709                                                                               -3.422                                      219     GLY N    9.670       38.554                                                                               -3.289                                      219     GLY C    7.570       37.384                                                                               -3.681                                      220     THR N    6.561       36.638                                                                               -3.205                                      220     THR C    4.879       37.044                                                                               -4.864                                      220     THR CB   4.825       34.819                                                                               -3.526                                      220     THR CG2  5.704       33.696                                                                               -2.900                                      221     SER CA   3.984       39.201                                                                               -5.169                                      221     SER O    4.117       40.208                                                                               -7.277                                      221     SER OG   3.435       40.282                                                                               -3.149                                      222     MET CE   6.471       42.771                                                                               -5.173                                      222     MET CG   8.506       41.399                                                                               -6.602                                      222     MET CA   6.916       39.670                                                                               -7.638                                      222     MET O    7.084       38.567                                                                               -9.775                                      223     ALA CA   6.469       36.020                                                                               -8.885                                      223     ALA O    5.133       35.948                                                                               -10.929                                     224     SER N    4.076       36.360                                                                               -9.038                                      224     SER C    2.661       37.161                                                                               -11.039                                     224     SER CB   1.801       36.995                                                                               -8.603                                      225     PRO N    3.156       38.411                                                                               -11.159                                     225     PRO C    3.764       38.469                                                                               -13.626                                     225     PRO CB   3.653       40.511                                                                               -12.054                                     225     PRO CD   3.735       39.224                                                                               -10.054                                     226     HIS CA   5.446       36.879                                                                               -14.362                                     226     HIS O    4.425       35.809                                                                               -16.293                                     226     HIS CG   7.814       36.859                                                                               -13.358                                     226     HIS CD2  8.883       37.118                                                                               -14.167                                     226     HIS NE2  9.771       37.866                                                                               -13.443                                     227     VAL CA   2.583       34.388                                                                               -14.727                                     227     VAL O    1.018       34.773                                                                               -16.490                                     227     VAL CG1  1.076       32.476                                                                               -14.246                                     228     ALA N    1.003       36.242                                                                               -14.814                                     228     ALA C    0.543       37.538                                                                               -16.868                                     228     ALA CB   -0.307      38.353                                                                               -14.668                                     229     GLY CA   2.352       38.408                                                                               -18.239                                     229     GLY O    2.189       37.375                                                                               -20.384                                     230     ALA CA   2.794       34.801                                                                               -19.546                                     230     ALA O    1.380       34.205                                                                               -21.343                                     231     ALA N    0.385       34.623                                                                               -19.328                                     231     ALA C    -1.256      35.423                                                                               -20.864                                     231     ALA CB   -1.932      34.664                                                                               -18.549                                     232     ALA CA   -1.013      37.663                                                                               -21.792                                     232     ALA O    -0.841      37.501                                                                               -24.187                                     233     LEU N    0.935       36.726                                                                               -22.967                                     233     LEU C    0.821       35.169                                                                               -24.880                                     233     LEU CB   3.063       35.877                                                                               -23.907                                     233     LEU CD1  5.259       36.342                                                                               -22.921                                     234     ILE N    0.357       34.199                                                                               -24.047                                     234     ILE CG1  0.454       31.223                                                                               -23.105                                     234     ILE CG2  -1.803      30.900                                                                               -24.091                                     234     ILE C    -1.621      33.597                                                                               -25.434                                     235     LEU N    -2.390      34.465                                                                               -24.779                                     235     LEU C    -3.258      35.843                                                                               -26.672                                     235     LEU CB   -4.432      35.765                                                                               -24.378                                     235     LEU CD1  -5.652      35.683                                                                               -22.145                                     236     SER N    -2.094      36.438                                                                               -26.798                                     236     SER C    -1.491      36.292                                                                               -29.144                                     236     SER CB   -0.633      38.234                                                                               -27.733                                     237     LYS N    -1.046      35.067                                                                               -28.882                                     237     LYS C    -2.113      33.277                                                                               -30.268                                     237     LYS CB   0.272       33.112                                                                               -29.551                                     237     LYS CD   2.020       31.535                                                                               -30.442                                     237     LYS CE   2.345       30.762                                                                               -31.7                                       238     HIS N    -2.951      32.989                                                                               -29.                                        238     HIS C    -5.334      32.599                                                                               -28.691                                     238     HIS CB   -3.948      30.862                                                                               -28.511                                     238     HIS ND1  -1.707      29.679                                                                               -28.835                                     238     HIS CE1  -1.086      28.851                                                                               -29.642                                     239     PRO N    -5.848      33.917                                                                               -29.365                                     239     PRO C    -8.204      34.052                                                                               -28.532                                     239     PRO CB   -7.018      35.977                                                                               -29.713                                     239     PRO CD   -5.436      34.439                                                                               -30.668                                     240     ASN CA   -9.529      32.041                                                                               -29.216                                     240     ASN O    -10.540     30.610                                                                               -27.576                                     240     ASN CG   -7.971      30.827                                                                               -30.889                                     240     ASN ND2  -7.670      29.509                                                                               -30.986                                     241     TRP CA   -8.304      30.124                                                                               -26.120                                     241     TRP O    -9.043      31.833                                                                               -24.686                                     241     TRP CG   -6.094      28.903                                                                               -26.557                                     241     TRP CD2  -4.839      28.324                                                                               -26.155                                     241     TRP CE2  -4.414      27.476                                                                               -27.216                                     241     TRP CZ2  -3.195      26.786                                                                               -27.174                                     241     TRP CH2  -2.470      26.873                                                                               -26.005                                     242     THR CA   -10.458     30.119                                                                               -22.911                                     242     THR O    -8.335      29.674                                                                               -21.937                                     242     THR OG1  -10.837     27.786                                                                               -22.476                                     243     ASN N    -9.946      30.659                                                                               -20.611                                     243     ASN OD1  -11.465     31.518                                                                               -16.768                                     243     ASN CB   -9.708      31.530                                                                               -18.332                                     243     ASN C    -8.657      29.303                                                                               -19.010                                     244     THR N    -9.564      28.362                                                                               -19.283                                     244     THR C    -8.133      26.393                                                                               -19.802                                     244     THR CB   -10.665     26.088                                                                               -19.494                                     244     THR CG2  -10.503     24.595                                                                               -19.158                                     245     GLN CA   -6.964      26.362                                                                               -21.962                                     245     GLN D    -4.573      26.393                                                                               -21.447                                     245     GLN CG   -8.265      25.526                                                                               -23.989                                     245     GLN OE1  -9.306      26.769                                                                               -25.727                                     246     VAL N    -5.697      28.304                                                                               -21.218                                     246     VAL C    -3.936      28.462                                                                               -19.467                                     246     VAL CB   -4.779      30.555                                                                               -20.621                                     246     VAL CG2  -5.169      31.138                                                                               -21.959                                     247     ARG CA   -4.380      27.714                                                                               -17.168                                     247     ARG O    -2.705      25.985                                                                               -16.764                                     247     ARG CG   -4.987      27.095                                                                               -14.852                                     247     ARG NE   -5.440      26.757                                                                               -12.546                                     247     ARG NH1  -7.064      27.484                                                                               -11.210                                     248     SER N    -4.480      25.505                                                                               -18.131                                     248     SER C    -2.657      24.086                                                                               -19.073                                     248     SER CB   -5.034      23.408                                                                               -19.372                                     249     SER N    -2.500      24.853                                                                               -20.136                                     249     SER C    -0.071      25.302                                                                               -19.940                                     249     SER CB   -1.369      25.758                                                                               -22.068                                     250     LEU N    -0.289      26.333                                                                               -19.160                                     250     LEU CD1  -0.373      30.453                                                                               -17.268                                     250     LEU CB   0.178       28.063                                                                               -17.505                                     250     LEU C    1.092       25.694                                                                               -17.265                                     251     GLN N    0.068       25.007                                                                               -16.714                                     251     GLN OE1  -2.819      23.424                                                                               -12.935                                     251     GLN CG   -1.218      24.814                                                                               -13.994                                     251     GLN CA   0.381       23.941                                                                               -15.745                                     251     GLN O    1.743       22.014                                                                               -15.616                                     252     ASN CA   1.082       21.206                                                                               -18.282                                     252     ASN O    2.809       20.442                                                                               -19.768                                     252     ASN CG   -1.036      19.926                                                                               -18.571                                     237     LYS NZ   3.525       29.848                                                                               -31.596                                     238     HIS CA   -4.168      32.163                                                                               -29.379                                     238     HIS O    -5.713      32.584                                                                               -27.562                                     238     HIS CG   -3.009      29.921                                                                               -29.237                                     238     HIS CD2  -3.137      29.258                                                                               -30.394                                     238     HIS NE2  -1.948      28.600                                                                               -30.599                                     239     PRO CA   -6.908      34.779                                                                               -28.773                                     239     PRO O    -8.949      34.519                                                                               -27.662                                     239     PRO CG   -6.666      35.294                                                                               -31.027                                     240     ASN N    -8.386      32.969                                                                               -29.227                                     240     ASN C    -9.508      31.180                                                                               -27.980                                     240     ASN CB   -9.403      31.249                                                                               -30.535                                     240     ASN OD1  -7.008      31.590                                                                               -31.147                                     241     TRP N    -8.354      31.006                                                                               -27.304                                     241     TRP C    -9.106      30.638                                                                               -24.936                                     241     TRP CB   -6.879      29.830                                                                               -25.679                                     241     TRP CD1  -6.338      28.433                                                                               -27.818                                     241     TRP NE1  -5.362      27.547                                                                               -28.211                                     241     TRP CE3  -4.097      28.406                                                                               -24.981                                     241     TRP CZ3  -2.912      27.667                                                                               -24.943                                     242     THR N    -9.727      29.781                                                                               -24.142                                     242     THR C    -9.469      30.176                                                                               -21.747                                     242     THR CB   -11.579     29.032                                                                               -22.675                                     242     THR CG2  -12.494     28.907                                                                               -23.895                                     243     ASN ND2  -11.787     30.404                                                                               -18.747                                     243     ASN CG   -11.093     31.131                                                                               -17.905                                     243     ASN CA   -9.053      30.731                                                                               -19.444                                     243     ASN O    -7.593      29.136                                                                               -18.440                                     244     THR CA   -9.381      26.934                                                                               -19.059                                     244     THR O    -7.324      25.757                                                                               -19.111                                     244     THR OG1  -11.735     26.675                                                                               -18.684                                     245     GLN N    -8.082      26.716                                                                               -21.073                                     245     GLN C    -5.647      27.020                                                                               -21.520                                     245     GLN CB   -7.330      26.599                                                                               -23.397                                     245     GLN CD   -8.493      25.873                                                                               -25.428                                     245     GLN NE2  -7.745      25.312                                                                               -26.370                                     246     VAL CA   -4.477      29.040                                                                               -20.770                                     246     VAL O    -2.705      28.227                                                                               -19.361                                     246     VAL CG1  -3.544      31.272                                                                               -20.027                                     247     ARG N    -4.767      28.240                                                                               -18.462                                     247     ARG C    -3.770      26.292                                                                               -17.340                                     247     ARG CB   -5.533      27.667                                                                               -16.149                                     247     ARG CD   -6.056      27.179                                                                               -13.793                                     247     ARG CZ   -5.893      26.866                                                                               -11.315                                     247     ARG NH2  -5.177      26.428                                                                               -10.270                                     248     SER CA   -4.039      24.131                                                                               -18.426                                     248     SER D    -1.848      23.253                                                                               -18.583                                     248     SER OG   -6.146      23.090                                                                               -18.532                                     249     SER CA   -1.223      24.874                                                                               -20.851                                     249     SER O    1.026       24.705                                                                               -20.049                                     249     SER OG   -0.300      25.419                                                                               -22.956                                     250     LEU CD2  1.824       29.814                                                                               -18.222                                     250     LEU CG   0.352       29.438                                                                               -18.151                                     250     LEU CA   0.718       26.837                                                                               -18.216                                     250     LEU O    2.283       25.421                                                                               -17.032                                     251     GLN NE2  -2.750      25.512                                                                               -12.237                                     251     GLN CD   -2.345      24.550                                                                               -13.034                                     251     GLN CB   -0.857      23.621                                                                               -14.877                                     251     GLN C    0.959       22.664                                                                               -16.361                                     252     ASN N    0.633       22.394                                                                               -17.590                                     252     ASN C    2.394       21.359                                                                               -18.991                                     252     ASN CB   0.004       20.780                                                                               -19.292                                     252     ASN OD1  -0.836      19.355                                                                               -17.502                                     252     ASN ND2  -2.234      19.834                                                                               -19.161                                     253     THR CA   4.256       22.717                                                                               -19.713                                     253     THR O    6.348       23.733                                                                               -19.427                                     253     THR OG1  3.595       24.957                                                                               -20.428                                     254     THR N    5.218       23.177                                                                               -17.551                                     254     THR C    7.466       22.700                                                                               -16.612                                     254     THR CB   5.664       23.558                                                                               -15.132                                     254     THR CG2  4.530       24.549                                                                               -14.802                                     255     THR CA   9.771       22.594                                                                               -15.817                                     255     THR O    9.439       22.786                                                                               -13.474                                     255     THR OG1  11.082      23.709                                                                               -17.321                                     256     LYS N    9.606       20.702                                                                               -14.314                                     256     LYS C    10.522      20.333                                                                               -12.063                                     256     LYS CB   9.024       18.590                                                                               -13.249                                     256     LYS CD   10.286      16.948                                                                               -11.777                                     256     LYS NZ   9.243       14.869                                                                               -11.054                                     257     LEU CA   11.272      21.038                                                                               -9.893                                      257     LEU D    12.096      20.565                                                                               -7.732                                      257     LEU CG   11.357      23.620                                                                               -10.568                                     257     LEU CD2  12.678      23.468                                                                               -11.325                                     258     GLY CA   10.602      18.793                                                                               -6.879                                      258     GLY D    8.283       18.956                                                                               -7.202                                      259     ASP CA   7.757       17.896                                                                               -4.516                                      259     ASP O    6.859       20.039                                                                               -4.214                                      259     ASP CG   6.781       17.128                                                                               -2.241                                      259     ASP OD2  7.098       16.299                                                                               -1.321                                      260     SER CA   4.481       19.587                                                                               -5.529                                      260     SER O    3.500       21.503                                                                               -4.446                                      260     SER OG   2.745       17.937                                                                               -5.448                                      261     PHE CA   3.831       20.468                                                                               -1.885                                      261     PHE O    3.944       22.848                                                                               -1.432                                      261     PHE CG   3.549       20.337                                                                               0.715                                       261     PHE CD2  4.401       21.060                                                                               1.558                                       261     PHE CE2  3.945       21.602                                                                               2.748                                       262     TYR N    5.778       21.758                                                                               -2.305                                      262     TYR C    6.820       23.689                                                                               -3.545                                      262     TYR CB   8.123       22.455                                                                               -1.851                                      262     TYR CD1  8.084       20.484                                                                               -0.364                                      262     TYP CE1  8.062       19.873                                                                               0.882                                       262     TYR CZ   8.069       20.672                                                                               2.018                                       263     TYR N    6.626       23.104                                                                               -4.693                                      263     TYR C    5.626       23.680                                                                               -6.956                                      263     TYR CB   7.928       22.768                                                                               -6.681                                      263     TYR CD1  10.064      24.046                                                                               -6.657                                      263     TYR CE1  11.335      24.328                                                                               -6.168                                      263     TYR CZ   11.838      23.618                                                                               -5.106                                      264     GLY N    4.471       23.161                                                                               -6.516                                      264     GLY C    3.847       22.196                                                                               -8.556                                      265     LYS N    3.436       22.477                                                                               -9.754                                      265     LYS C    5.188       22.232                                                                               -11.464                                     265     LYS CB   2.755       22.071                                                                               -12.044                                     265     LYS CD   0.710       20.548                                                                               -12.079                                     265     LYS NZ   -1.678      20.757                                                                               -12.489                                     266     GLY CA   7.120       23.612                                                                               -11.325                                     266     GLY O    6.177       25.793                                                                               -11.648                                     267     LEU CA   8.490       26.660                                                                               -13.097                                     267     LEU O    7.953       25.909                                                                               -15.298                                     267     LEU CG   10.432      28.060                                                                               -14.058                                     267     LEU CD2  11.924      27.921                                                                               -14.327                                     268     ILE CA   6.406       28.035                                                                               -15.944                                     268     ILE O    8.539       28.793                                                                               -16.912                                     268     ILE CG1  6.099       30.541                                                                               -15.592                                     268     ILE CD1  5.399       31.769                                                                               -16.262                                     253     THR N    3.018       22.505                                                                               -18.923                                     253     THR C    5.381       23.247                                                                               -18.818                                     253     THR CB   4.086       23.672                                                                               -20.952                                     253     THR CG2  3.147       23.130                                                                               -22.032                                     254     THR CA   6.216       23.612                                                                               -16.588                                     254     THR O    7.402       21.580                                                                               -17.095                                     254     THR OG1  5.129       22.178                                                                               -15.040                                     255     THR N    8.499       23.296                                                                               -16.076                                     255     THR C    9.621       22.031                                                                               -14.414                                     255     THR CB   11.080      23.455                                                                               -15.897                                     255     THR CG2  12.286      22.628                                                                               -15.406                                     256     LYS CA   9.364       20.063                                                                               -13.010                                     256     LYS O    11.662      20.274                                                                               -12.592                                     256     LYS CG   9.018       17.805                                                                               -11.921                                     256     LYS CE   10.212      15.940                                                                               -10.623                                     257     LEU N    10.212      20.674                                                                               -10.824                                     257     LEU C    11.250      20.232                                                                               -8.614                                      257     LEU CB   11.187      22.547                                                                               -9.522                                      257     LEU CD1  11.245      25.003                                                                               -9.921                                      258     GLY N    10.431      19.282                                                                               -8.298                                      258     GLY C    9.168       18.703                                                                               -6.373                                      259     ASP N    9.024       18.282                                                                               -5.150                                      259     ASP C    6.659       18.941                                                                               -4.709                                      259     ASP CB   7.996       17.540                                                                               -3.053                                      259     ASP OD1  5.611       17.527                                                                               -2.354                                      260     SER N    5.560       18.610                                                                               -5.312                                      260     SER C    4.046       20.362                                                                               -4.289                                      260     SER CB   3.345       18.919                                                                               -6.289                                      261     PHE N    4.241       19.778                                                                               -3.112                                      261     PHE C    4.544       21.846                                                                               -1.863                                      261     PHE CB   4.053       19.749                                                                               -0.563                                      261     PHE CD1  2.206       20.163                                                                               1.125                                       261     PHE CE1  1.737       20.717                                                                               2.315                                       261     PHE CZ   2.605       21.465                                                                               3.114                                       262     TYR CA   6.688       22.914                                                                               -2.251                                      262     TYR O    7.201       24.853                                                                               -3.393                                      262     TYR CG   8.146       21.892                                                                               -0.454                                      262     TYR CD2  8.149       22.669                                                                               0.698                                       262     TYR CE2  8.114       22.069                                                                               1.962                                       262     TYR OH   7.965       20.029                                                                               3.205                                       263     TYR CA   6.812       23.655                                                                               -6.022                                      263     TYP O    5.781       24.117                                                                               -8.111                                      263     TYR CG   9.279       23.035                                                                               -6.068                                      263     TYR CD2  9.800       22.342                                                                               -4.995                                      263     TYR CE2  11.062      22.640                                                                               -4.491                                      263     TYR OH   13.065      23.949                                                                               -4.597                                      264     GLY CA   3.301       23.064                                                                               -7.412                                      264     GLY O    4.647       21.274                                                                               -8.365                                      265     LYS CA   3.834       21.798                                                                               -10.971                                     265     LYS O    5.684       21.563                                                                               -12.386                                     265     LYS CG   1.490       21.563                                                                               -11.305                                     265     LYS CE   -0.692      20.496                                                                               -11.391                                     266     GLY N    5.787       23.226                                                                               -10.817                                     266     GLY C    7.155       25.052                                                                               -11.818                                     267     LEU N    8.262       25.336                                                                               -12.480                                     267     LEU C    7.804       26.771                                                                               -14.437                                     267     LEU CB   10.010      26.855                                                                               -13.214                                     267     LEU CD1  10.096      29.331                                                                               -13.250                                     268     ILE N    7.064       27.863                                                                               -14.632                                     268     ILE C    7.426       28.246                                                                               -17.065                                     268     ILE CB   5.369       29.210                                                                               -15.899                                     268     ILE CG2  4.243       28.925                                                                               -14.867                                     269     ASN N    7.007       27.843                                                                               -18.237                                     269     ASN CA   7.802       27.975                                                                               -19.                                        269     ASN O    5.965       27.760                                                                               -20.                                        269     ASN CG   9.161       26.806                                                                               -21.210                                     269     ASN ND2  10.011      25.796                                                                               -21.472                                     270     VAL CA   5.863       30.418                                                                               -21.614                                     270     VAL O    5.097       29.969                                                                               -23.872                                     270     VAL CG1  6.849       32.757                                                                               -21.879                                     271     GLN N    7.325       29.701                                                                               -23.352                                     271     GLN C    6.869       27.934                                                                               -25.031                                     271     GLN CB   9.104       29.220                                                                               -24.964                                     271     GLN CD   10.901      28.585                                                                               -26.582                                     271     GLN NE2  11.702      28.553                                                                               -25.510                                     272     ALA CA   6.224       25.712                                                                               -24.240                                     272     ALA O    3.898       25.505                                                                               -25.001                                     273     ALA N    4.247       26.661                                                                               -23.135                                     273     ALA C    2.081       27.528                                                                               -24.020                                     273     ALA CB   2.736       27.773                                                                               -21.585                                     274     ALA CB   2.952       30.391                                                                               -26.210                                     274     ALA C    1.730       28.367                                                                               -27.090                                     275     GLN N    2.350       27.194                                                                               -27.314                                     275     GLN C    2.147       27.261                                                                               -29.777                                     275     GLN OT   1.193       27.361                                                                               -30.590                                     275     GLN CG   0.501       24.664                                                                               -27.447                                     275     GLN DE1  -1.376      23.895                                                                               -28.729                                     269     ASN C    6.839       28.554                                                                               -20.485                                     269     ASN CB   8.432       26.653                                                                               -19.895                                     269     ASN OD1  8.993       27.626                                                                               -22.122                                     270     VAL N    6.908       29.868                                                                               -20.724                                     270     VAL C    6.059       30.007                                                                               -23.054                                     270     VAL CB   5.656       31.950                                                                               -21.422                                     270     VAL CG2  4.420       32.362                                                                               -22.232                                     271     GLN CA   7.603       29.270                                                                               -24.744                                     271     GLN O    6.213       27.806                                                                               -26.091                                     271     GLN CG   9.406       28.618                                                                               -26.338                                     271     GLN OE1  11.369      28.579                                                                               -27.718                                     272     ALA N    6.977       26.999                                                                               -24.092                                     272     ALA C    4.701       25.958                                                                               -24.164                                     272     ALA CB   6.743       24.742                                                                               -23.172                                     273     ALA CA   2.840       26.921                                                                               -22.859                                     273     ALA O    0.899       27.219                                                                               -24.255                                     274     ALA N    2.755       28.404                                                                               -24.762                                     274     ALA CA   2.109       29.144                                                                               -25.847                                     274     ALA O    0.980       28.949                                                                               -27.921                                     275     GLN CA   2.048       26.389                                                                               -28.527                                     275     GLN O    3.260       27.807                                                                               -29.916                                     275     GLN CB   0.666       25.734                                                                               -28.520                                     275     GLN CD   -0.823      23.936                                                                               -27.631                                     275     GLN NE2  -1.373      23.411                                                                               -26.538                                     ______________________________________                                    

The above structural studies together with the above referenced kinetic data and kinetic data presented herein indicate that the subsites in the binding cleft of subtilisin are capable of interacting with substrate amino acid residues from P-4 to P-2'.

The most extensively studied of the above residues are Gly166, Gly169 and Ala152. There amino acids were identified as residues within the S-1 subsite. As seen in FIG. 3, which is a stereoview of the S-1 subsite, Gly166 and Gly169 occupy positions at the bottom of the S-1 subsite, whereas Ala152 occupies a position near the top of S-1, close to the catalytic Ser221.

All 19 amino acid substitutions of Gly166 and Gly169 have been made. As will be indicated in the examples which follow, the preferred replacement amino acids for Gly166 and/or Gly169 will depend on the specific amino acid occupying the P-1 position of a given substrate.

The only substitutions of Ala152 presently made and analyzed comprise the replacement of Ala152 with Gly and Ser. The results of these substitutions on P-1 specificity will be presented in the examples.

In addition to those residues specifically associated with specificity for the P-1 substrate amino acid, Tyr104 has been identified as being involved with P-4 specificity. Substitutions at Phe189 and Tyr217, however, are expected to respectively effect P-2' and P-1' specificity.

The catalytic activity of subtilisin has also been modified by single amino acid substitutions at Asn155.

The catalytic triad of subtilisin is shown in FIG. 4. As can be seen, Ser221, His64 and Asp32 are positioned to facilitate nucleophilic attack by the serine hydoxylate on the carbonyl of the scissile peptide bond. Several hydrogen bonds may also help to stabilize the transition state complex for the tetrahedral substrate intermediate. One hydrogen bond is between aspartate and the positively charged histidine, ND1. Kossiakoff, A. A., et al. (1981) Biochem. 20, 6462-5474. A second hydrogen bond forms between the scissile amide nitrogen of the substrate and the (NE2) proton on the histidine. A third set of hydrogen bonds forms between the enzyme and the oxyanion that is produced from the carbonyl oxygen of the substrate. This latter set of hydrogen bonds is formed differently by the mammalian serine proteases and substilisin. A fourth hydrogen bond appears to exist between the amide nitrogen of the peptide bond between P-1 and P-2 and the carbonyl oxygen of Ser125. Specifically, x-ray crystallographic studies of chymotrypsin (Henderson, R. (1970) J. Mol. Biol. 54, 341) indicate that two hydrogen bonds form between the substrate oxyanion and two main-chain amide protons from the enzyme (Gly193 and the catalytic Ser195). Crystallographic studies of subtilisin (Robertus, et al. (1972) Biochem. 11, 4293-4303; Matthews, et al. (1975) J. Biol. Chem. 250, 7120-7126; Poulos, et al. (1976) J. Biol. Chem. 250, 1097-1103) show that two hydrogen bonds are also formed with the oxyanion; one hydrogen bond donor is from the catalytic serine-221 main-chain amide while the other is from one of the NE2 protons of the asparagine-155 side chain. See FIG. 4.

Asn155 was substituted with Ala, Asp, His, Glu and Thr. These substitutions were made to investigate the the stabilization of the charged tetrahedral intermediate of the transition state complex by the potential hydrogen bond between the side chain of Asn155 and the oxyanion of the intermediate. These particular substitutions caused large decreases in substrate turnover, kcat (200 to 4,000 fold), marginal decreases in substrate binding K_(M) (up to 7 fold), and a loss in transition state stabilization energy of 2.2 to 4.7 kcal/mol. The retention of K_(M) and the drop in kcat will make these mutant enzymes useful as binding proteins for specific peptide sequences, the nature of which will be determined by the specificity of the precursor protease.

Various other amino acid residues have been identified which affect alkaline stability. In some cases, mutants having altered alkaline stability also have altered thermal stability.

In B amyloliquefaciens subtilisin residues Asp36, Ile107, Lys170, Ser204 and Lys213 have been identified as residues which upon substitution with a different amino acid alter the alkaline stability of the mutated enzyme as compared to the precursor enzyme. The substitution of Asp36 with Ala and the substitution of Lys170 with Glu each resulted in a mutant enzyme having a lower alkaline stability as compared to the wild type subtilisin. When Ile107 was substituted with Val, Ser204 substituted with Cys, Arg or Leu or Lys213 substituted with Arg, the mutant subtilisin had a greater alkaline stability as compared to the wild type subtilisin. However, the mutant Ser204P demonstrated a decrease in alkaline stability.

In addition, other residues, previously identified as being associated with the modification of other properties of subtilisin, also affect alkaline stability. These residues include Ser24, Met50, Glu156, Gly166, Gly169 and Tyr217. Specifically the following particular substitutions result in an increased alkaline stability: Ser24C, Met50F, Gly156Q or S, Gly166A, H, K, N or Q, Gly169S or A, and Tyr217F, K, R or L. The mutant Met50V, on the other hand, results in a decrease in the alkaline stability of the mutant subtilisin as compared to wild type subtilisin.

Other residues involved in alkaline stability based on the alkaline stability screen include the mutants of Table I for residues Asp197 and Met222.

Various other residues have been identified as being involved in thermal stability as determined by the thermal stability screen herein. These residues include the above identified residues which affect alkaline stability and Met199 and Tyr21. These latter two residues are also believed to be important for alkaline stability. Mutants at these residues include I199 and F21.

The amino acid sequence of B. amyloliquefaciens substilisin has also been modified by substituting two or more amino acids of the wild-type sequence. Six categories of multiply substituted mutant subtilisin have been identified. The first two categories comprise thermally and oxidatively stable mutants. The next three other categories comprise mutants which combine the useful properties of any of several single mutations of B. amyloliquefaciens subtilisin. The last category comprises mutants which have modified alkaline and/or thermal stability.

The first category is double mutants in which two cysteine residues have been substituted at various amino acid residue positions within the subtilisin BPN' molecule. Formation of disulfide bridges between the two substituted cysteine residues results in mutant subtilisins with altered thermal stability and catalytic activity. These mutants include A21/C22/C87 and C24/C87 which will be described in more detail in Example 11.

The second category of multiple subtilisin mutants comprises mutants which are stable in the presence of various oxidizing agents such as hydrogen peroxide or peracids. Examples 1 and 2 describe these mutants which include F50/Il24/Q222, F50/Il124, F50/Q222, F50/L124/Q222, I124/Q222 and L124/Q222.

The third category of multiple subtilisin mutants comprises mutants with substitutions at position 222 combined with various substitutions at positions 166 or 169. These mutants, for example, combine the property of oxidative stability of the A222 mutation with the altered substrate specificity of the various 166 or 169 substitutions. Such multiple mutants include A166/A222, A166/C222, F166/C222, K166/A222, K166/C222, V166/A222 and V166/C222. The K166/A222 mutant subtilisin, for example, has a kcat/K_(M) ratio which is approximately two times greater than that of the single A222 mutant subtilisin when compared using a substrate with phenylalanine as the P-1 amino acid. This category of multiple mutant is described in more detail in Example 12.

The fourth category of multiple mutants combines substitutions at position 156 (Glu to Q or S) with the substitution of Lys at position 166. Either of these single mutations improve enzyme performance upon substrates with glutamate as the P-1 amino acid. When these single mutations are combined, the resulting multiple enzyme mutants perform better than either precursor. See Example 9.

The fifth category of multiple mutants contain the substitution of up to four amino acids of the B. amyloliquefaciens subtilisin sequence. These mutants have specific properties which are virtually identical to the properties of the subtilisin from B. licheniformis. The subtilisin from B. licheniformis differs from B. amyloliquefaciens subtilisin at 87 out of 275 amino acids. The multiple mutant F50/S156/A169/L217 was found to have similar substrate specificity and kinetics to the licheniformis enzyme. (See Example 13.) However, this is probably due to only three of the mutations (S156, A169 and L217) which are present in the substrate binding region of the enzyme. It is quite surprising that, by making only three changes out of the 87 different amino acids between the sequence of the two enzymes, the B. amyloliquifaciens enzyme was converted into an enzyme with properties similar to B. licheniformis enzyme. Other enzymes in this series include F50/Q156/N166/L217 and F50/S156/L217.

The sixth category of multiple mutants includes the combination of substitutions at position 107 (Ile to V) with the substitution of Lys at position 213 with Arg, and the combination of substitutions of position 204 (preferably Ser to C or L but also to all other amino acids) with the substituion of Lys at position 213 with R. Other multiple mutants which have altered alkaline stability include Q156/K166, Q156/N166, S156/K166, S156/N166 (previously identified as having altered substrate specificity), and F50/S156/A169/L217 (previously identified as a mutant of B. amyloliquifaciens subtilisin having properties similar to subtilisin from B. licheniformis). The mutant F50/V107/R213 was constructed based on the observed increase in alkaline stability for the single mutants F50, V107 and R213. It was determined that the V107/R213 mutant had an increased alkaline stability as compared to the wild type subtilisin. In this particular mutant, the increased alkaline stability was the result of the cumulative stability of each of the individual mutations. Similarly, the mutant F50/V107/R213 had an even greater alkaline stability as compared to the V107/R213 mutant indicating that the increase in the alkaline stability due to the F50 mutation was also cumulative.

Table IV summarizes the multiple mutants which have been made including those not mentioned above.

In addition, based in part on the above results, substitution at the following residues in subtilisin is expected to produce a multiple mutant having increased thermal and alkaline stability: Ser24, Met50, Ile107, Glu156, Gly166, Gly169, Ser204, Lys213, Gly215, and Tyr217.

                  TABLE IV                                                         ______________________________________                                                      Triple, Quadruple                                                 Double Mutants                                                                              or Other Multiple                                                 ______________________________________                                         C22/C87      F50/I124/Q222                                                     C24/C87      F50/L124/Q222                                                     V45/V48      F50/L124/A222                                                     C49/C94      A21/C22/C87                                                       C49/95       F50/S156/N166/L217                                                C50/C95      F50/Q156/N166/L217                                                C50/C110     F50/S156/A169/L217                                                F50/I124     F50/S156/L217                                                     F50/Q222     F50/Q156/K166/L217                                                I124/Q222    F50/Q156/K166/K217                                                Q156/D166    F50/Q156/K166/K217                                                Q156/K166    F50/S156/K166/K217                                                Q156/N166    F50/V107/R213                                                     S156/D166    [S153/S156/A158/G159/S160/Δ161-                             S156/K166    164/I165/S166/A169/R170]                                          S156/N166    L204/R213                                                         S156/A169    R213/204A, E, Q, D, N, G, K,                                      A166/A222    V, R, T, P, I, M, F, Y, W                                         A166/C222    or H                                                              F166/A222    V107/R213                                                         F166/C222                                                                      K166/A222                                                                      K166/C222                                                                      V166/A222                                                                      V166/C222                                                                      A169/A222                                                                      A169/A222                                                                      A169/C222                                                                      A21/C22                                                                        ______________________________________                                    

In addition to the above identified amino acid residues, other amino acid residues of subtilisin are also considered to be important with regard to substrate specificity. These are the aforementioned residues which have yet to be mutated. Mutation of each of these residues is expected to produce changes in the substrate specificity of subtilisin. Moreover, multiple mutations among these residues and among the previously identified residues are also expected to produce subtilisin mutants having novel substrate specificity.

Particularly important residues are His67, Ile107, Leu126 and Leu135. Mutation of His67 should alter the S-1' subsite, thereby altering the specificity of the mutant for the P-1' substrate residue. Changes at this position could also affect the pH activity profile of the mutant. This residue was identified based on the inventor's substrate modeling from product inhibitor complexes.

Ile107 is involved in P-4 binding. Mutation at this position thus should alter specificity for the P-4 substrate residue. Ile107 was also identified by molecular modeling from product inhibitor complexes.

The S-2 binding site includes the Leu126 residue. Modification at this position should therefore affect P-2 specificity. Moreover, this residue is believed to be important to convert subtilisin to an amino peptidase. The pH activity profile should also be modified by appropriate substitution. These residues were identified from inspection of the refined model, the three dimensional structure from modeling studies. A longer side chain is expected to preclude binding of any side chain at the S-2 subsite. Therefore, binding would be restricted to subsites S-1, S-1', S-2', S-3' and cleavage would be forced to occur after the amino terminal peptide.

Leu135 is in the S-4 subsite and if mutated should alter substrate specificity for P-4 if mutated. This residue was identified by inspection of the three-dimensional structure and modeling based on the product inhibitor complex of F222.

In addition to these sites, specific amino acid residues within the segments 97-103, 126-129 and 213-215 are also believed to be important to substrate binding.

Segments 97-103 and 126-129 form an antiparallel beta sheet with the main chain of substrate residues P-4 through P-2. Mutating residues in those regions should affect the substrate orientation through main chain (enzyme)--main chain (substrate) interactions, since the main chain of these substrate residues do not interact with these particular residues within the S-4 through S-2 subsites. Within the segment 97-103, Gly97 and Asp99 may be mutated to alter the position of residues 101-103 within the segment. Changes at these sites must be compatible, however. In B. amyloliquifaciens subtilisin Asp99 stabilizes a turn in the main chain tertiary folding that affects the direction of residues 101-103. B. licheniformis subtilisin Asp97, functions in an analogous manner.

In addition to Gly97 and Asp99, Ser101 interacts with Asp99 in B. amyloliquefaciens subtilisin to stabilize the same main chain turn. Alterations at this residue should alter the 101-103 main chain direction. Mutations at Glu103 are also expected to affect the 101-103 main chain direction.

The side chain of Gly102 interacts with the substrate P-3 amino acid. Side chains of substituted amino acids thus are expected to significantly affect specificity for the P-3 substrate amino acids.

All the amino acids within the 127-129 segment are considered important to substrate specificity. Gly 127 is positioned such that its side chain interacts with the S-1 and S-3 subsites. Altering this residue thus should alter the specificity for P-1 and P-3 residues of the substrate.

The side chain of Gly128 comprises a part of both the S-2 and S-4 subsites. Altered specificity for P-2 and P-4 therefore would be expected upon mutation. Moreover, such mutation may convert subtilisin into an amino peptidase for the same reasons substitutions of Leu126 would be expected to produce that result. The Pro129 residue is likely to restrict the conformational freedom of the sequence 126-133, residues which may play a major role in determining P-1 specificity. Replacing Pro may introduce more flexibility thereby broadening the range of binding capabilities of such mutants.

The side chain of Lys213 is located within the S-3 subsite. All of the amino acids within the 213-215 segment are also considered to be important to substrate specificity. Accordingly, altered P-3 substrate specificity is expected upon mutation of this residue.

The Tyr214 residue does not interact with substrate but is positioned such that it could affect the conformation of the hair pin loop 204-217.

Finally, mutation of the Gly215 residue should affect the S-3' subsite, and thereby alter P-3' specificity.

In addition to the above substitutions of amino acids, the insertion or deletion of one or more amino acids within the external loop comprising residues 152-172 may also affect specificity. This is because these residues may play a role in the "secondary contact region" described in the model of streptomyces subtilisin inhibitor complexed with subtilisin. Hirono, et al. (1984) J. Mol. Biol. 178, 389-413. Thermitase K has a deletion in this region, which eliminates several of these "secondary contact" residues. In particular, deletion of residues 161 through 164 is expected to produce a mutant subtilisin having modified substrate specificity. In addition, a rearrangement in this area induced by the deletion should alter the position of many residues involved in substrate binding, predominantly at P-1. This, in turn, should affect overall activity against proteinaceous substrates.

The effect of deletion of residues 161 through 164 has been shown by comparing the activity of the wild type (WT) enzyme with a mutant enzyme containing this deletion as well as multiple substitutions (i.e., S153/S156/A158/G159/S160/A161-164/I165/S166/A169/R170). This produced the following results:

                  TABLE V                                                          ______________________________________                                                 kcat       K.sub.M kcat/K.sub.M                                        ______________________________________                                         WT        50           1.4e-4  3.6e5                                           Deletion  8            5.0e-6  1.6e6                                           mutant                                                                         ______________________________________                                    

The WT has a kcat 6 times greater than the deletion mutant but substrate binding is 28 fold tighter by the deletion mutant. The overall efficiency of the deletion mutant is thus 4.4 times higher than the WT enzyme.

All of these above identified residues which have yet to be substituted, deleted or inserted into are presented in Table VI.

                  TABLE VI                                                         ______________________________________                                         Substitution/Insertion/Deletion                                                Residues                                                                       ______________________________________                                                 His67        Ala152                                                            Leu126       Ala153                                                            Leu135       Gly154                                                            Gly97        Asn155                                                            Asp99        Gly156                                                            Ser101       Gly157                                                            Gly102       Gly160                                                            Glu103       Thr158                                                            Leu126       Ser159                                                            Gly127       Ser161                                                            Gly128       Ser162                                                            Pro129       Ser163                                                            Tyr214       Thr164                                                            Gly215       Val165                                                                         Gly166                                                                         Tyr167                                                                         Pro168                                                                         Gly169                                                                         Lys170                                                                         Tyr171                                                                         Pro172                                                    ______________________________________                                    

The mutants herein may be obtained as salts. It is clear that the ionization state of a protein will be dependent on the pH of the surrounding medium, if it is in solution, or of the solution from which it is prepared, if it is in solid form. Acidic proteins are commonly prepared as, for example, the ammonium, sodium, or potassium salts; basic proteins as the chlorides, sulfates, or phosphates. Accordingly, the present application includes both electrically neutral and salt forms of the designated carbonyl hydrolases, and the term carbonyl hydrolase refers to the organic structural backbone regardless of ionization state.

The carbonyl hydrolase mutants are particularly useful in the food processing and cleaning arts. The carbonyl hydrolases, including mutants, are produced by fermentation as described herein and recovered by suitable techniques. See for example K. Anstrup, 1974, Industrial Aspects of Biochemistry, ed. B. Spencer pp. 23-46. They are formulated with detergents or other surfactants in accord with methods known per se for use in industrial processes, especially laundry. In the latter case the enzymes are combined with detergents, builders, bleach and/or flourescent whitening agents as is known in the art for proteolytic enzymes. Suitable detergents include linear alkyl benzene sulfonates, alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linear alcohol. The compositions may be formulated in granular or liquid form. See for example U.S. Pat. Nos. 3,623,957; 4,404,128; 4,381,247; 4,404,115; 4,318,818; 4,261,868; 4,242,219; 4,142,999; 4,111,855; 4,011,169; 4,090,973; 3,985,686; 3,790,482; 3,749,671; 3,560,392; 3,558,498; and 3,557,002.

The following disclosure is intended to serve as a representation of embodiments herein, and should not be construed as limiting the scope of this application. These specific examples disclose the construction of certain of the above identified mutants. The construction of the other mutants, however, is apparent from the disclosure herein and that presented in EPO Publication No. 0130756.

Glossary of Experimental Manipulations

In order to simplify the Examples certain frequently occurring methods will be referenced by shorthand phrases.

Plasmids are designated by a small p proceeded and/or followed by capital letters and/or numbers. The starting plasmids herein are commercially available, are available on an unrestricted basis, or can be constructed from such available plasmids in accord with published procedures.

"Klenow treatment" refers to the process of filling a recessed 3' end of double stranded DNA with deoxyribonucleotides complementary to the nucleotides making up the protruding 5' end of the DNA strand or exonucleolytic removal of a protruding 3' end of a double stranded DNA fragment. This process is usually used to fill in a recessed end resulting from a restriction enzyme cleavage of DNA. This creates a blunt or flush end, as may be required for further ligations. Treatment with Klenow is accomplished by reacting (generally for 15 minutes at 15° C.) the appropriate complementary deoxyribonucleotides with the DNA to be filled in under the catalytic activity (usually 10 units) of the Klenow fragment of E. coli DNA polymerase I ("Klenow"). Klenow and the other reagents needed are commercially available. The procedure has been published extensively. See for example T. Maniatis, et al., 1982, Molecular Cloning, pp. 107-108.

"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific is called a restriction site. "Partial" digestion refers to incomplete digestion by restriction enzyme, i.e., conditions are chosen that result in cleavage of some but not all of the sites for a given restriction endonuclease in a DNA substrate. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements as established by the enzyme suppliers were used. Restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters and then, generally, a number representing the microorganism from which each restriction enzyme originally was obtained. In general, about 1 mg of plasmid or DNA fragment is used with about 1 unit of enzyme in about 20 μl of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After incubation, protein is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme infrequently is followed with bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to prevent the two restriction cleaved ends of a DNA fragment from "circularizing" or forming a closed loop that would impede insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5' terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional (T. Maniatis, et al., Id., pp. 133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the digest on 6 percent polyacrylamide gel electrophoresis, identification of the fragment of interest by molecular weight (using DNA fragments of known molecular weight as markers), removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example, see R. Lawn, et al., 1981, "Nucleic Acids Res." 9:6103-6114, and D. Goeddel, et al., 1980, "Nucleic Acids Res." 8:4057.

"Southern Analysis" is a method by which the presence of DNA sequences in a digest or DNA-containing composition is confirmed by hybridization to a known, labelled oligonucleotide or DNA fragment. For the purposes herein, Southern analysis shall mean separation of digests on 1 percent agarose and depurination as described by G. Wahl, et al., 1979, "Proc. Nat. Acad. Sci. U.S.A." 76:3683-3687, transfer to nitrocellulose by the method of E. Southern, 1975, "J. Mol. Biol." 98:503-517, and hybridization as described by T. Maniatis, et al., 1978, "cell" 15:687-701.

"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integrant. Unless otherwise stated, the method used herein for transformation of E. coli is the CaCl₂ method of Mandel, et al., 1970, "J. Mol. Biol." 53:154, and for Bacillus, the method of Anagnostopolous, et al., 1961, "J. Bact." 81:741-746.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (T. Maniatis, et al., Id., p. 146). Unless otherwise stated, ligation was accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated. Plasmids from the transformants were prepared, analyzed by restriction mapping and/or sequenced by the method of Messing, et al., 1981, "Nucleic Acids Res.", 9:309.

"Preparation" of DNA from transformants means isolating plasmid DNA from microbial culture. Unless otherwise stated, the alkaline/SDS method of Maniatis, et al., Id., p. 90, was used.

"Oligonucleotides" are short length single or double stranded polydeoxynucleotides which were chemically synthesized by the method of Crea, et al., 1980,

"Nucleic Acids Res." 8:2331-2348 (except that mesitylene nitrotriazole was used as a condensing agent) and then purified on polyacrylamide gels. All mutant plasmids were transformed (Anagnostopoulos, C., et al. (1961) J. Bacteriol. 81, 741-746) into BG2036 (Yang, M. (1984) J. Bacteriol. 160, 15-21) to express mutant subtilisins as described (Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521). All literature citations are expressly incorporated by reference. The following is presented by way of example and is not to be construed as a limitation to the scope of the invention.

EXAMPLE 1 Identification of Peracid Oxidizable Residues of Subtilisin Q222 and L222

The activity of naturally-occurring subtilisin is rapidly reduced up to 85% by a series of oxidants. One of the most characterized modification is the conversion of Met222 to met-sulfoxide in the presence of hydrogen peroxide. Stauffer, C. E., et al. (1969) J. Biol. Chem. 244, 5333-5338. This defect has been eliminated by substituting a variety of non-oxidizable amino acids into this position by site-directed mutagenesis of the B. amyloliquifaciens enzyme, thereby confering enhanced stability to hydrogen peroxide. See EPO Publication No. 0130756 and Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518. However, as shown in FIGS. 6A and 6B, organic peracid oxidants can still inactivate the mutant enzymes Met222L and Met222Q (L222 and Q222). This example describes the identification of peracid oxidizable sites in 222 substituted mutant subtilisins.

The first step was to determine the type of amino acid involved in peracid oxidation. Except under drastic conditions (Means, G. E., et al. (1971) Chemical Modifications of Proteins, Holden-Day, S.F., Calif., pp. 160-162), organic peracids modify only methionine and tryptophan in subtilisin. In order to rule out tryptophan as a candidate, difference spectra of the enzyme over the 250 nm to 350 nm range were determined during an inactivation titration employing the reagent, diperdodecanoic acid (DPDA) as the oxidant. Despite quantitative inactivation of the enzyme, no change in absorbance over this wavelength range was noted as shown in FIGS. 7A and 7B. Oxidation of tryptophan would be expected to result in marked changes over this region. Fontana, A., et al. (1980) Methods in Peptide and Protein Sequence Analysis (C. Birr ed.) Elsevier, N.Y., p. 309. The absence of tryptophan modification implied oxidation of one or more of the remaining methionines of B. amyloliquefaciens subtilisin. See FIGS. 1A and 1B.

To confirm this result the recombinant subtilisin Met222F was cleaved with cyanogen bromide (CNBr) both before and after oxidation by DPDA. The peptides produced by CNBr cleavage were analyzed on high resolution SDS-pyridine peptide gels (SPG).

Subtilisin Met222F (F222) was oxidized in the following manner. Purified F222 was resuspended in 0.1 M sodium borate pH 9.5 at 10 mg/ml and was added to a final concentration of 26 diperdodecanoic acid (DPDA) at 26 mg/ml was added to produce an effective active oxygen concentration of 30 ppm. The sample was incubated for at least 30 minutes at room temperature and then quenched with 0.1 volume of 1 M Tris pH 8.6 buffer to produce a final concentration of 0.1 M Tris pH 8.6). 3 mM phenylmethylsulfonyl fluoride (PMSF) was added and 2.5 ml of the sample was applied to a Pharmacia PD10 column equilibrated in 10 mM sodium phosphate pH 6.2, 1 mM PMSF. 3.5 ml of 10 mM sodium phosphate pH6.2, 1 mM PMSF was applied and the eluant collected. The sample was assumed to be at 7 mg/ml based on the observation that a 2.5 ml sample of untreated F222 at 10 mg/ml in phosphate buffer when treated with PMSF and desalted in the same manner on a Pharmacia PD10 column produced a concentration of about 7 mg/ml.

F222 and DPDA oxidized F222 were precipitated with 9 volumes of acetone at -20° C. For 100 ug of protein, the acetone mixture was vortexed and centrifuged in a Fischer tabletop centrifuge for 10 minutes. The pellets were washed once with 0.5 ml acetone and then dried. The sample was carefully resuspended at 10 mg/ml in 8M urea in 88% formic acid and allowed to sit for 5 minutes. An equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein) and the samples incubated for 2 hours at room temperature in the dark. Prior to gel electrophoresis, the samples were lyophilized for 3-4 hours in a Spin Vac (Savant Instruments) and the pellets were resuspended at 2-5 mg/ml in sample buffer (1% pyridine, 5% NaDodSO₄, 5% glycerol and bromophenol blue) and disassociated at 95° C. for 3 minutes.

The samples were electrophoresed on discontinuous polyacrylamide gels as described by Kyte and Rodriguez (Kyte, J., et al. (1983) Anal. Bioch. 133, 515-522). The gels were stained using the Pharmacia silver staining technique (Sammons, D. W., et al. (1981) Electrophoresis 2 135-141).

The results of this experiment are shown in FIG. 8. As can be seen, F222 treated with CNBr only gives nine resolved bands on SPG. However, when F222 is also treated with DPDA prior to cleavage, bands X, 7 and 9 disappear whereas bands 5 and 6 are greatly increased in intensity.

In order to determine which of the methionines were effected, each of the CNBr peptides was isolated by reversed phase HPLC and further characterized. The buffer system in both Solvent A (aqueous) and Solvent B (organic) for all HPLC separations was 0.05% TEA-TFA. Solutions were prepared by adding equal volumes of neat triethylamine and neat trifloroacetic acid to the solvent. Programs for the gradients were generated on a Waters Systems Controller. In all cases unless noted, solvent A consisted of 0.05% TEA-TFA in H₂ O, solvent B was 0.05% TEA-TFA in 1-propanol, and the flow rate was 0.5 ml/minute. For HPLC analysis, two injections of 1 mg enzyme digest were used. Three samples were acetone precipitated, washed and dried as above. The dried 1 mg samples were resuspended at 10 mg/ml in 8M urea, 88% formic acid; an equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein). After incubation for 2 hours in the dark at room temperature, the samples were desalted on a 0.8 cm×7 cm column of Tris Acryl GF05 coarse resin (IBF, Paris, France) equilibrated with 40% solvent B, 60% solvent A. 200 ul samples were applied at a flow rate of 1 ml a minute and 1.0-1.2 ml collected by monitoring the absorbance at 280 nm. Prior to injection on the HPLC, each desalted sample was diluted with 3 volumes of solvent A. The samples were injected at 1.0 ml/min (2 minutes) and the flow then adjusted to 0.5 ml/min (100% A). After 2 minutes, a linear gradient to 60% B at 1.0% B/min was initiated. From each 1 mg run, the pooled peaks were sampled (50 ul) and analyzed by gel electrophoresis as described above.

Each polypeptide isolated by reversed phase HPLC was further analyzed for homogeneity by SPG. The position of each peptide on the known gene sequence (Wells, J. A., et al. (1983) Nucleic Acids Res. 11 7911-7924) was obtained through a combination of amino acid compositional analysis and, where needed, amino terminal sequencing. Prior to such analysis the following peptides were to rechromatographed.

1. CNBr peptides from F222 not treated with DPDA:

Peptide 5 was subjected to two additional reversed phase separations. The 10 cm C4 column was equilibrated to 80%A/20%B and the pooled sample applied and washed for 2 minutes. Next an 0.5% ml B/min gradient was initiated. Fractions from this separation were again rerun, this time on the 25 cm C4 column, and employing 0.05% TEA-TFA in acetonitrile/1-propanol (1:1) for solvent B. The gradient was identical to the one just described.

Peptide "X" was subjected to one additional separation after the initial chromatography. The sample was applied and washed for 2 minutes at 0.5 ml/min (100%A), and a 0.5% ml B/min gradient was initiated.

Peptides 7 and 9 were rechromatographed in a similar manner to the first rerun of peptide 5.

Peptide 8 was purified to homogeneity after the initial separation.

2. CNBr Peptides from DPDA Oxidized F222:

Peptides 5 and 6 from a CNBr digest of the oxidized F222 were purified in the same manner as peptide 5 from the untreated enzyme.

Amino acid compositional analysis was obtained as follows. Samples (˜nM each amino acid) were dried, hydrolyzed in vacuo with 100 ul 6N HCl at 106° C. for 24 hours and then dried in a Speed Vac. The samples were analyzed on a Beckmann 6300 AA analyzer employing ninhydrin detection.

Amino terminal sequence data was obtained as previously described (Rodriguez, H., et al. (1984) Anal. Biochem. 134, 538-547).

The results are shown in Table VII and FIG. 9.

                  TABLE VII                                                        ______________________________________                                         Amino and COOH terminii of CNBr fragments                                      Terminus and Method                                                            Fragment    amino, method  COOH, method                                        ______________________________________                                         X            1, sequence    50, composition                                    9            51, sequence  119, composition                                    7           125, sequence  199, composition                                    8           200, sequence  275, composition                                    5ox          1, sequence   119, composition                                    6ox         120, composition                                                                              199, composition                                    ______________________________________                                    

Peptides 50x and 60x refer to peptides 5 and 6 isolated from CNBr digests of the oxidized protein where their respective levels are enhanced. From the data in Table VII and the comparison of SPG tracks for the oxidized and native protein digests in FIG. 8, it is apparent that (1) Met50 is oxidized leading to the loss of peptides X and 9 and the appearance of 5; and (2) Met124 is also oxidized leading to the loss of peptide 7 and the accumulation of peptide 6. Thus oxidation of B. amyloliquefaciens subtilisin with the peracid, diperdocecanoic acid leads to the specific oxidation of methionines 50 and 124.

EXAMPLE 2 Substitution at Met50 and Met124 in Subtilisin Met222Q

The choice of amino acid for substitution at Met50 was based on the available sequence data for subtilisins from B. licheniformis (Smith, E. C., et al. (1968) J. Biol. Chem. 243, 2184-2191), B.DY (Nedkov, P., et al. (1983) Hoppe Sayler's Z. Physiol. Chem. 364 1537-1540), B. amylosacchariticus (Markland, F. S., et al. (1967) J. Biol. Chem. 242 5198-5211) and B. subtilis (Stahl, M. L., et al. (1984) J. Bacteriol. 158, 411-418). In all cases, position 50 is a phenylalanine. See FIG. 5A-1, 5A-2, 5B-1, 5B-2 and 5C. Therefore, Phe50 was chosen for construction.

At position 124, all known subtilisins possess a methionine. See FIGS. 5A-1, 5A-2, 5B-1, 5B-2, and 5C. Molecular modelling of the x-ray derived protein structure was therefore required to determine the most probable candidates for substitution. From all 19 candidates, isoleucine and leucine were chosen as the best residues to employ. In order to test whether or not modification at one site but not both was sufficient to increase oxidative stability, all possible combinations were built on the Q222 backbone (F50/Q222, I124/Q222, F50/I124/Q222).

A. Construction of Mutations Between Codons 45 and 50

All manipulations for cassette mutagenesis were carried out on pS4.5 using methods disclosed in EPO Publication No. 0130756 and Wells, J. A., et al, (1985) Gene 34, 315-323. The pΔ50 in FIG. 10, line 4, mutations was produced using the mutagenesis primer shown in FIG. 10, line 6, and employed an approach designated as restriction-purification which is described below. Wells, J. A., et al. (1986) Phil. Trans. R. Soc. Lond. A (in press). Briefly, a M13 template containing the subtilisin gene, M13mp11-SUBT was used for heteroduplex synthesis (Adelman, et al (1983), DNA 2, 183-193). Following transfection of JM101 (ATCC 33876), the 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was subcloned from M13mp11 SUBT rf into a recipient vector fragment of pBS42 the construction of which is described in EPO Publication No. 0130756. To enrich for the mutant sequence (pΔ50, line 4), the resulting plasmid pool was digested with KpnI, and linear molecules were purified by polyacrylamide gel electrophoresis. Linear molecules were ligated back to a circular form, and transformed into E. coli MM294 cells (ATCC 31446). Isolated plasmids were screened by restriction analysis for the KpnI site. KpnI⁺ plasmids were sequenced and confirmed the pΔ50 sequence. Asterisks in FIG. 11 indicate the bases that are mutated from the wid type sequence (line 4). pΔ50 (line 4) was cut with Stul and EcoRI and the 0.5 Kb fragment containing the 5' half of the subtilisin gene was purified (fragment 1). pΔ50 (line 4) was digested with KpnI and EcoRI and the 4.0 Kb fragment containing the 3' half of the subtilisin gene and vector sequences was purified (fragment 2). Fragments 1 and 2 (line 5), and duplex DNA cassettes coding for mutations desired (shaded sequence, line 6) were mixed in a molar ratio of 1:1:10, respectively. For the particular construction of this example the DNA cassette contained the triplet TTT for codon 50 which encodes Phe. This plasmid was designated pF-50. The mutant subtilisin was designated F-50.

B. Construction of Mutation Between Codons 122 and 127

The procedure of Example 2A was followed in substantial detail except that the mutagenesis primer of FIG. 11, line 7 was used and restriction-purification for the EcoRV site in pΔ124 was used. In addition, the DNA cassette (shaded sequence, FIG. 11, line 6) contained the triplet ATT for codon 124 which encodes Ile and CTT for Leu. Those plasmids which contained the substitution of Ile for Met124 were designeated pI124. The mutant subtilisin was designated I124.

C. Construction of Various F50/I124/Q222 Multiple Mutants

The triple mutant, F50/I124/Q222, was constructed from a three-way ligation in which each fragment contained one of the three mutations. The single mutant Q222 (pQ222) was prepared by cassette mutagenesis as described in EPO Publication No. 0130756. The F50 mutation was contained on a 2.2 kb AvaII to PvuII fragment from pF50; the I124 mutation was contained on a 260 bp PvuII to AvaII fragment from pI124; and the Q222 mutation was contained on 2.7 kb AvaII to AvaII fragment from pQ222. The three fragments were ligated together and transformed into E. coli MM294 cells. Restriction analysis of plasmids from isolated transformants confirmed the construction. To analyze the final construction it was convenient that the AvaII site at position 798 in the wild-type subtilisin gene was eliminated by the I124 construction. The F50/Q222 and I124/Q222 mutants were constructed in a similar manner except that the appropriate fragment from pS4.5 was used for the final construction.

D. Oxidative Stability of Q222 Mutants

The above mutants were analyzed for stability to peracid oxidation. As shown in FIG. 12, upon incubation with diperdodecanoic acid (protein 2 mg/mL, oxidant 75 ppm[0]), both the I124/Q222 and the F50/I124/Q222 are completely stable whereas the F50/Q222 and the Q222 are inactivated. This indicates that conversion of M124 to I124 in subtilisin Q222 is sufficient to confer resistance to organic peracid oxidants.

EXAMPLE 3 Subtilisin Mutants Having Altered Substrate Specificity-Hydrophobic Substitutions at Residues 166

Subtilisin contains an extended binding cleft which is hydrophobic in character. A conserved glycine at residue 166 was replaced with twelve non-ionic amino acids which can project their side-chains into the S-1 subsite. These mutants were constructed to determine the effect of changes in size and hydrophobicity on the binding of various substrates.

A. Kinetics for Hydrolysis of Substrates Having Altered P-1 Amino Acids by Subtilisin BPN' from B. Amyloliquefaciens

Wild-type subtilisin was purified from B. subtilis culture supernatants expressing the B. amyloliquefaciens subtilisin gene (Wells, J. A., et al. (1983) Nucleic Acids Res. 11, 7911-7925) as previously described (Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521). Details of the synthesis of tetrapeptide substrates having the form succinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide (where X is the P1 amino acid) are described by DelMar, E. G., et al. (1979) Anal. Biochem. 99, 316-320. Kinetic parameters, K_(M) (M) and kcat(s⁻¹) were measured using a modified progress curve analysis (Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521). Briefly, plots of rate versus product concentration were fit to the differential form of the rate equation using a non-linear regression algorithm. Errors in kcat and K_(M) for all values reported are less than five percent. The various substrates in Table VIII are ranged in order of decreasing hydrophobicity. Nozaki, Y. (1971), J. Biol. Chem. 246, 2211-2217; Tanford C. (1978) Science 200, 1012).

                  TABLE VIII                                                       ______________________________________                                         P1 substrate                    kcat/K.sub.M                                   Amino Acid                                                                               kcat(S.sup.-1)                                                                             1/K.sub.M (M.sup.-1)                                                                     (s.sup.-1 M - 1)                               ______________________________________                                         Phe       50          7,100     360,000                                        Tyr       28          40,000    1,100,000                                      Leu       24          3,100     75,000                                         Met       13          9,400     120,000                                        His       7.9         1,600     13,000                                         Ala       1.9         5,500     11,000                                         Gly       0.003       8,300     21                                             Gln       3.2         2,200     7,100                                          Ser       2.8         1,500     4,200                                          Glu       0.54        32        16                                             ______________________________________                                    

The ratio of kcat/K_(M) (also referred to as catalytic efficiency) is the apparent second order rate constant for the conversion of free enzyme plus substrate (E+S) to enzyme plus products (E+P) (Jencks, W. P., Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287). The log (kcat/K_(M)) is proportional to transition state binding energy, ΔG_(T). A plot of the log kcat/K_(M) versus the hydrophobicity of the P1 side-chain (FIG. 14) shows a strong correlation (r=0.98), with the exception of the glycine substrate which shows evidence for non-productive binding. These data show that relative differences between transition-state binding energies can be accounted for by differences in P-1 side-chain hydrophobicity. When the transition-state binding energies are calculated for these substrates and plotted versus their respective side-chain bydrophobicities, the line slope is 1.2 (not shown). A slope greater than unity, as is also the case for chymotrypsin (Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Harper, J. W., et al. (1984) Biochemistry, 23, 2995-3002), suggests that the P1 binding cleft is more hydrophobic than ethanol or dioxane solvents that were used to empirically determine the hydrophobicity of amino acids (Nozaki, Y., et al. J. Biol. Chem. (1971) 246, 2211-2217; Tanford, C. (1978) Science 200, 1012).

For amide hydrolysis by subtilisin BPN', kcat can be interpreted as the acylation rate constant and K_(M) as the dissociation constant, for the Michaelis complex (E·S), Ks. Gutfreund, H., et al (1956) Biochem. J. 63, 656. The fact that the log kcat, as well as log 1/K_(M), correlates with substrate hydrophobicity is consistent with proposals (Robertus, J. D., et al. (1972) Biochemistry 11, 2439-2449; Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303) that during the acylation step the P-1 side-chain moves deeper into the hydrophobic cleft as the substrate advances from the Michaelis complex (E·S) to the tetrahedral transition-state complex (E·S). However, these data can also be interpreted as the hydrophobicity of the P1 side-chain effecting the orientation, and thus the susceptibility of the scissile peptide bond to nucleophilic attack by the hydroxyl group of the catalytic Ser221.

The dependence of kcat/K_(M) on P-1 side chain hydrophobicity suggested that the kcat/K_(M) for hydrophobic substrates may be increased by increasing the hydrophobicity of the S-1 binding subsite. To test this hypothesis, hydrophobic amino acid substitutions of Gly166 were produced.

Since hydrophobicity of aliphatic side-chains is directly proportional to side-chain surface area (Rose, G. D., et al. (1985) Science 229, 834-838; Reynolds, J. A., et al. (1974) Proc. Natl. Acad. Sci. USA 71, 2825-2927), increasing the hydrophobicity in the S-1 subsite may also sterically hinder binding of larger substrates. Because of difficulties in predicting the relative importance of these two opposing effects, we elected to generate twelve non-charged mutations at position 166 to determine the resulting specificities against non-charged substrates of varied size and hydrophobicity.

B. Cassette Mutagenesis of the P1 Binding Cleft

The preparation of mutant subtilisims containing the substitution of the hydrophobic amino acids Ala, Val and Phe into residue 166 has been described in EPO Publication No. 0130756. The same method was used to produce the remaining hydrophobic mutants at residue 166. In applying this method, two unique and silent restriction sites were introduced in the subtilisin genes to closely flank the target codon 166. As can be seen in FIG. 13, the wild type sequence (line 1) was altered by site-directed mutagenesis in M13 using the indicated 37mer mutagenesis primer, to introduce a 13 bp delection (dashedline) and unique SacI and XmaI sites (underlined sequences) that closely flank codon 166. The subtilisin gene fragment was subcloned back into the E. coli--B. subtilis shuttle plasmid, pBS42, giving the plasmid pΔ66 (FIG. 13, line 2). pΔ166 was cut open with SacI and XmaI, and gapped linear molecules were purified (FIG. 13, line 3). Pools of synthetic oligonucleotides containing the mutation of interest were annealed to give duplex DNA cassettes that were ligated into gapped pΔ166 (underlined and overlined sequences in FIG. 13, line 4). This construction restored the coding sequence except over position 166 (NNN; line 4). Mutant sequences were confirmed by dideoxy sequencing. Asterisks denote sequence changes from the wild type sequence. Plasmids containing each mutant B. amyloliquefaciens subtilisin gene were expressed at roughly equivalent levels in a protease deficient strain of B. subtilis, BG2036 as previously described. EPO Publication No. 0130756; Yang, M., et al. (1984) J. Bacteriol. 160, 15-21; Estell, D. A., et al (1985) J. Biol. Chem. 260, 6518-6521.

C. Narrowing Substrate Specificity by Steric Hindrance

To probe the change in substrate specificity caused by steric alterations in the S-1 subsite, position 166 mutants were kinetically analyzed versus P1 substrates of increasing size (i.e., Ala, Met, Phe and Tyr). Ratios of kcat/K_(M) are presented in log form in FIGS. 15A and 15B to allow direct comparisons of transitionstate binding energies between various enzymesubstrate pairs.

According to transition state theory, the free enery difference between the free enzyme plus substrate (E+S) and the transition state complex (E·S.sup.≠) can be calculated from equation (1),

    .sup.Δ G.sup.≠.sub.T =-RT 1n kcat/K.sub.M +RT1n kT/h(1)

in which kcat is the turnover number, K_(M) is the Michaelis constant, R is the gas constant, T is the temperature, k is Boltzmann's constant, and h is Planck's constant. Specificity differences are ezpressed quantitatively as differences between transition state binding energies (i.e., ΔΔG.sup.≠_(t)), and can be calculated from equation (2).

    .sup.ΔΔ G.sup.≠.sub.T =-RT 1n(kcat/K.sub.M).sub.A /(kcat/Km).sub.B                                          (2)

A and B represent either two different substrates assayed againt the same enzyme, or two mutant enzymes assayed against the same substrate.

As can be seen from FIG. 15A, as the size of the side-chain at position 166 increases the substrate preference shifts from large to small P-1 side-chains. Enlarging the side-chain at position 166 causes kcat/K_(M) to decrease in proportion to the size of the P-1 substrate side-chain (e.g., from Gly166 (wild-type) through W166, the kcat/K_(M) for the Tyr substrate is decreased most followed in order by the Phe, Met and Ala P-1 substrates).

Specific steric changes in the position 166 side-chain, such as he presence of a β-hydroxyl group, β- or γ-aliphatic branching, cause large decreases in kcat/K_(M) for larger P1 substrates. Introducing a β-hydroxyl group in going from Δ166 (FIGS. 15A and 15B) to S166 (FIG. 15B), causes an 8 fold and 4 fold reduction in kcat/K_(M) for Phe and Tyr substrates, respectively, while the values for Ala and Met substrates are unchanged. Producing a β-branched structure, in going from S166 to T166, results in a drop of 14 and 4 fold in kcat/K_(M) for Phe and Tyr, respectively. These differences are slightly magnified for V166 which is slightly larger and isosteric with T166. Enlarging the β-branched substituents from V166 to I166 causes a lowering of kcat/K_(M) between two and six fold toward Met, Phe and Tyr substrates. Inserting a γ-branched structure, by replacing M166 (FIG. 15A) with L166 (FIG. 15B), produces a 5 fold and 18 fold decrease in kcat/K_(M) for Phe and Tyr substrates, respectively. Aliphatic γ-branched appears to induce less steric hindrance toward the Phe P-1 substrate than β-branching, as evidenced by the 100 fold decrease in kcat/K_(M) for the Phe substrate in going from L166 to I166.

Reductions in kcat/K_(M) resulting from increases in side chain size in the S-1 subsite, or specific structural features such as β- and γ-branching, are quantitatively illustrated in FIGS. 16A, 16B, 16C and 16D. The kcat/K_(M) values for the position 166 mutants determined for the Ala, Met, Phe, and Tyr P-1 substrates (top panel through bottom panel, respectively), are plotted versus the position 166 side-chain volumes (Chothia, C. (1984) Ann. Rev. Biochem. 53, 537-572). Catalytic efficiency for the Ala substrate reaches a maximum for I166, and for the Met substrate it reaches a maximum between V166 and L166. The Phe substrate shows a broad kcat/K_(M) peak but is optimal with A166. Here, the β-branched position 166 substitutions form a line that is parallel to, but roughly 50 fold lower in kcat/K_(M) than side-chains of similar size [i.e., C166 versus T166, L166 versus I166]. The Tyr substrate is most efficiently utilized by wild type enzyme (Gly166), and there is a steady decrease as one proceeds to large position 166 side-chains. The β-branched and γ-branched substitutions form a parallel line below the other non-charged substitutions of similar molecular volume.

The optimal substitution at position 166 decreases in volume with increasing volume of the P1 substrate [i.e., I166/Ala substrate, L166/Met substrate, A166/Phe substrate, Gly166/Tyr substrate]. The combined volumes for these optimal pairs may approximate the volume for productive binding in the S-1 subsite. For the optimal pairs, Gly166/Tyr substrate, A166/Phe substrate, L166/Met substrate, V166/Met substrate, and I166/Ala substrate, the combined volumes are 266,295,313,339 and 261 A³, respectively. Subtracting the volume of the peptide backbone from each pair (i.e., two times the volume of glycine), an average side-chain volume of 160=32A³ for productive binding can be calculated.

The effect of volume, in excess to the productive binding volume, on the drop in transition-state binding energy can be estimated from the Tyr substrate curve (bottom panel, FIGS. 16A, 16B, 16C and 16D), because these data, and modeling studies (FIG. 2), suggest that any substitution beyond glycine causes steric repulsion. A best-fit line drawn to all the data (r=0.87) gives a slope indicating a loss of roughly 3 kcal/mol in transition state binding energy per 100A³ of excess volume. (100A³ is approximately the size of a leucyl side-chain.)

D. Enhanced Catalytic Efficiency Correlates with Increasing Hydrophobicity of the Position 166 Substitution

Substantial increases in kcat/K_(M) occur with enlargement of the position 166 side-chain, except for the Tyr P-1 substrate (FIGS. 16A, 16B, 16C and 16D). For example, kcat/K_(M) increases in progressing from Gly166 to I166 for the Ala substrate (net of ten-fold), from Gly166 to L166 for the Met substrate (net of ten-fold) and from Gly166 to A166 for the Phe substrate (net of two-fold). The increases in kcat/K_(M) cannot be entirely explained by the attractive terms in the van der Waals potential energy function because of their strong distance dependence (1/r⁶) and because of the weak nature of these attractive forces (Jencks, W. P., Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Levitt, M. (1976) J. Mol. Biol. 104, 59-107). For example, Levitt (Levitt, M. (1976) J. Mol. Biol. 104, 59-107) has calculated that the van der Waals attraction between two methionyl residues would produce a maximal interaction energy of roughly -0.2 kcal/mol. This energy would translate to only 1.4 fold increase in kcat/K_(M).

The increases of catalytic efficiency caused by side-chain substitutions at position 166 are better accounted for by increases in the hydrophobicity of the S-1 subsite. The increase kcat/K_(M) observed for the Ala and Met substrates with increasing position 166 side-chain size would be expected, because hydrophobicity is roughly proportional to side-chain surface area (Rose, G. D., et al. (1985) Science 229, 834-838; Reynolds, J. A., et al. (1974) Proc. Natl. Acad. Sci. USA 71, 2825-2927).

Another example that can be interpreted as a hydrophobic effect is seen when comparing kcat/K_(M) for isosteric substitutions that differ in hydrophobicity such as S166 and C166 (FIG. 16). Cysteine is considerably more hydrophobic than serine (-1.0 versus +0.3 kcal/mol) (Nozaki, Y., et al. (1971) J. Biol. Chem. 246, 2211-2217; Tanford, C. (1978) Science 200, 1012). The difference in hydrophobicity correlates with the observation that C166 becomes more efficient relative to Ser166 as the hydrophobicity of the substrates increases (i.e., Ala<Met<Tye<Phe). Steric hindrance cannot explain these differences because serine is considerably smaller than cysteine (99 versus 118A³). Paul, I. C., Chemistry of the -SH Group (ed. S. Patai, Wiley Interscience, New York, 1974) pp. 111-149.

E. Production of an Elastase-Like Specificity in Subtilisin

The I166 mutation illustrates particularly well that large changes in specificity can be produced by altering the structure and hydrophobicity of the S-1 subsite by a single mutation (FIG. 17). Progressing through the small hydrophobic substrates, a maximal specificity improvement over wild type occurs for the Val substrate (16 fold in kcat/K_(M)). As the substrate side chain size increases, these enhancements shrink to near unity (i.e., Leu and His substrates). The I166 enzyme becomes poorer against larger aromatic substrates of increasing size (e.g., I166 is over 1,000 fold worse against the Tyr substrate than is Gly166). We interpret the increase in catalytic efficiency toward the small hydrophobic substrates for I166 compared to Gly166 to the greater hydrophobicity of isoluecine (i.e., -1.8 kcal/mol versus 0). Nozaki, Y., et al. (1971) J. Biol. Chem. 246, 2211-2217; Tanford, C. (1978) Science 200, 1012. The decrease in catalytic efficiency toward the very large substrates for I166 versus Gly166 is attributed to steric repulsion.

The specificity differences between Gly166 and I166 are similar to the specificity differences between chymotrypsin and the evolutionary relative, elastase (Harper, J. W., et al (1984) Biochemistry 23, 2995-3002). In elastase, the bulky amino acids, Thr and Val, block access to the P-1 binding site for large hydrophobic substrates that are preferred by chymotrypsin. In addition, the catalytic efficiencies toward small hydrophobic substrates are greater for elastase than for chymotrypsin as we observe for 1166 versus Gly166 in subtilisin.

EXAMPLE 4 Substitution of Ionic Amino Acids for Gly166

The construction of subtilisin mutants containing the substitution of the ionic amino acids Asp, Asn, Gln, Lys and Arg are disclosed in EPO Publication No. 0130756. In addition, a limited analysis of substrate specificity was presented therein. The present example describes the construction of the mutant subtilisin containing Glu at position 166 (E166) and presents some of the specificity data on these mutants. Further data on position 166 and 156 single and double mutants will be presented infra.

pΔ66, described in Example 3, was digested with SacI and XmaI. The double strand DNA cassette (underlined and overlined) of line 4 in FIG. 13 contained the triplet GAA for the codon 166 to encode the replacement of Glu for Gly166. This mutant plasmid designated pQ166 was propagated in BG2036 as described. This mutant subtilisin, together with the other mutants containing ionic substituent amino acids at residue 166, were isolated as described and further analyzed for variations in substrate specificity.

Each of these mutants was analyzed with the tetrapeptide substrates, succinyl-L-AlaL-AlaProL-X-p-nitroanilide, where X was Phe, Ala and Glu.

The results of this analysis are shown in Table IX.

                  TABLE IX                                                         ______________________________________                                                  P-1 Substrate                                                                  (kcat/K.sub.M × 10.sup.-4)                                      Position 166                                                                              Phe          Ala    Glu                                             ______________________________________                                         Gly (wild type)                                                                           36.0         1.4    0.002                                           Asp (D)    0.5          0.4    <0.001                                          Glu (E)    3.5          0.4    <0.001                                          Asn (N)    18.0         1.2    0.004                                           Gln (Q)    57.0         2.6    0.002                                           Lys (K)    52.0         2.8    1.2                                             Arg (R)    42.0         5.0    0.08                                            ______________________________________                                    

These results indicate that charged amino acid substitutions at Gly166 have improved catalytic efficiencies (kcat/K_(M)) for oppositely charged P-1 substrates (as much as 500 fold) and poorer catalytic efficiency for like charged P-1 substrates.

EXAMPLE 5 Substitution of Glycine at Position 169

The substitution of Gly169 in B. amyloliquefaciens subtilisin with Ala and Ser is described in EPO Publication No. 0130756. The same method was used to make the remaining 17 mutants containing all other substituent amino acids for position 169.

The construction protocol is summarized in FIG. 18. The overscored and underscored double stranded DNA cassettes used contained the following triplet encoding the substitution of the indicated amino acid at residue 169.

    ______________________________________                                                  GCT          A                                                                 TGT          C                                                                 GAT          D                                                                 GAA          E                                                                 TTC          F                                                                 GGC          G                                                                 CAC          H                                                                 ATC          I                                                                 AAA          K                                                                 CTT          L                                                                 ATG          M                                                                 AAC          N                                                                 CCT          P                                                                 CAA          Q                                                                 AGA          R                                                                 AGC          S                                                                 ACA          T                                                                 GTT          V                                                                 TGG          W                                                                 TAC          Y                                                        ______________________________________                                    

Each of the plasmids containing a substituted Gly169 was designated pX169, where X represents the substituent amino acid. The mutant subtilisins were simialrly designated.

Two of the above mutant subtilisins, A169 and S169, were analyzed for substrate specificity against synthetic substrates containing Phe, Leu, Ala and Arg in the P-1 position. The following results are shown in Table X.

                  TABLE X                                                          ______________________________________                                         Effect of Serine and Alanine Mutations                                         at Position 169 on P-1 Substrate Specificity                                              P-1 Substrate (kcat/K.sub.M × 10.sup.-4)                      Position 169 Phe    Leu        Ala  Arg                                        ______________________________________                                         Gly (wild type)                                                                             40     10         1    0.4                                        A169         120    20         1    0.9                                        S169         50     10         1    0.6                                        ______________________________________                                    

These results indicate that substitutions of Ala and Ser at Gly169 have remarkably similar catalytic efficiencies against a range of P-1 substrates compared to their position 166 counterparts. This is probably because position 169 is at the bottom of the P-1 specificity subsite.

EXAMPLE 6 Substitution at Position 104

Tyr104 has been substituted with Ala, His, Leu, Met and Ser. The method used was a modification of the site directed mutagenesis method. According to the protocol of FIG. 19, a primer (shaded in line 4) introduced a unique HindIII site and a frame shift mutation at codon 104. Restriction-purification for the unique HindIII site facilitated the isolation of the mutant sequence (line 4). Restriction-selection against this HindIII site using pimers in line 5 was used to obtain position 104 mutants.

The following triplets were used in the primers of FIG. 19, line 5 for the 104 codon which substituted the following amino acids.

    ______________________________________                                         GCT         Ala        TTC        Phe                                          ATG         Met        CCT        Pro                                          CTT         Leu        ACA        Thr                                          AGC         Ser        TGG        Trp                                          CAC         His        TAC        Tyr                                          CAA         Gln        GTT        Val                                          GAA         Glu        AGA        Arg                                          GGC         Gly        AAC        Asn                                          ATC         Ile        GAT        Asp                                          AAA         Lys        TGT        Cys                                          ______________________________________                                    

The following substrates were used to analyze the substrate specificity of these mutants to give the indicated results in Table XI.

                  TABLE XI                                                         ______________________________________                                                 kcat     K.sub.M      Kcat/K.sub.M                                     Substrate WT     H104    WT    H104   WT   H104                                ______________________________________                                         sAAPFpNA  50.0   22.0    1.4e - 4                                                                             7.1e - 4                                                                              3.6e5                                                                               3.1e4                               sAAPApNA  3.2    2.0     2.3e - 4                                                                             1.9e - 3                                                                              1.4e4                                                                                 1e3                               sFAPFpNA  26.0   38.0    1.8e - 4                                                                             4.1e - 4                                                                              1.5e5                                                                               9.1e4                               sFAPApNA  0.32   2.4     7.3e - 5                                                                             1.4e - 4                                                                              4.4e3                                                                               1.6e4                               ______________________________________                                    

From these data it is clear that the substitution of His for Tyr at position 104 produces an enzyme which is more efficient (higher kcat/K_(M)) when Phe is at the P-4 substrate position than when Ala is at the P-4 substrate position.

EXAMPLE 7 Substitution of Ala152

Ala152 has been substituted by Gly and Ser to determine the effect of such substitutions on substrate specificity.

The wild type DNA sequence was mutated by the V152/P153 primer (FIG. 20, line 4) using the above restriction-purification approach for the new KPnI site. Other mutant primers (shaded sequences FIG. 20; S152, line 5 and G152, line 6) mutated the new KpnI site away and such mutants were isolated using the restriction-selection procedure as described above for loss of the KpnI site.

The results of these substitutions for the above synthetic substrates containing the P-1 amino acids Phe, Leu and Ala are shown in Table XII.

                  TABLE XII                                                        ______________________________________                                                   P-1 Substrate                                                                  (kcat/K.sub.M × 10.sup.-4)                                     Position 152                                                                               Phe          Leu    Ala                                            ______________________________________                                         Gly (G)     0.2          0.4    <0.04                                          Ala (wild type)                                                                            40.0         10.0   1.0                                            Ser (S)     1.0          0.5    0.2                                            ______________________________________                                    

These results indicate that, in contrast to positions 166 and 169, replacement of Ala152 with Ser or Gly causes a dramatic reduction in catalytic efficiencies across all substrates tested. This suggests Ala152, at the top of the S-1 subsite, may be the optimal amino acid because Ser and Gly are homologous Ala substitutes.

EXAMPLE 8 Substitution at Position 156

Mutants containing the substitution of Ser and Gln for Glu156 have been constructed according to the overall method depicted in FIG. 21. This method was designed to facilitate the construciton of multiple mutants at position 156 and 166 as will be described hereinafter. However, by regenerating the wild type Gly166, single mutations at Glu156 were obtained.

The plasmid pΔ166 is already depicted in line 2 of FIG. 13. The synthetic oligonucleotides at the top right of FIG. 21 represent the same DNA cassettes depicted in line 4 of FIG. 13. The plasmid p166 in FIG. 21 thus represents the mutant plasmids of Examples 3 and 4. In this particular example, p166 contains the wild type Gly166.

Construction of position 156 single mutants were prepared by ligation of the three fragments (1-3) indicated at the bottom of FIG. 21. Fragment 3, containing the carboxy-terminal portion of the subtilisin gene including the wild type position 166 codon, was isolated as a 610 bp SacI-BamHI fragment. Fragment 1 contained the vector sequences, as well as the amino-terminal sequences of the subtilisin gene through codon 151. To produce fragment 1, a unique KpnI site at codon 152 was introduced into the wild type subtilisin sequence from pS4.5. Site-directed mutagenesis in M13 employed a primer having the sequence 5'-TA-GTC-GTT-GCG-GTA-CCC-GGT-AAC-GAA-3' to produce the mutation. Enrichment for the mutant sequence was accomplished by restriction with KpnI, purification and self ligation. The mutant sequence containing the KpnI site was confirmed by direct plasmid sequencing to give pV-152. pV-152 (˜1 μg) was digested with KpnI and treated with 2 units of DNA polymerase I large fragment (Klenow fragment from Boeringer-Mannheim) plus 50 μM deoxynucleotide triphosphates at 37° C. for 30 min. This created a blunt end that terminated with codon 151. The DNA was extracted with 1:1 volumes phenol and CHCl₃ and DNA in the aqueous phase was precipitated by addition of 0.1 volumes SM ammonium acetate and two volumes ethanol. After centrifugation and washing the DNA pellet with 70% ethanol, the DNA was lyophilized. DNA was digested with BamHI and the 4.6 kb piece (fragment 1) was purified by acrylamide gel electrophoresis followed by electroelution. Fragment 2 was a duplex synthetic DNA cassette which when ligated with fragments 1 and 3 properly restored the coding sequence except at codon 156. The top strand was synthesized to contain a glutamine codon, and the complementary bottom strand coded for serine at 156. Ligation of heterophosphorylated cassettes leads to a large and favorable bias for the phosphorylated over the non-phosphorylated oligonucleotide sequence in the final segrated plasmid product. Therefore, to obtain Q156 the top strand was phosphorylated, and annealed to the non-phosphorylated bottom strand prior to ligation. Similarly, to obtain S156 the bottom strand was phosphorylated and annealed to the non-phosphorylated top strand. Mutant sequences were isolated after ligation and transformation, and were confirmed by restriction analysis and DNA sequencing as before. To express variant subtilisins, plasmids were transformed into a subtilisin-neutral protease deletion mutant of B. subtilis, BG2036, as previously described. Cultures were fermented in shake flasks for 24 h at 37° C. in LB media containing 12.5 mg/mL chloraphenicol and subtilisin was purified from culture supernatants as described. Purity of subtilisin was greater than 95% as judged by SDS PAGE. These mutant plasmids designated pS156 and pQ156 and mutant subtilisins designated S156 and Q156 were analyzed with the above synthetic substrates where P-1 comprised the amino acids Glu, Gln, Met and Lys. The results of this analyses are presented in Example 9.

EXAMPLE 9 Multiple Mutants With Altered Substrate Specificity--Substitution at Positions 156 and 166

Single substitutions of position 166 are described in Examples 3 and 4. Example 8 describes single substitutions at position 156 as well as the protocol of FIG. 21 whereby various double mutants comprising the substitution of various amino acids at positions 156 and 166 can be made. This example describes the construction and substrate specificity of subtilisin containing substitutions at position 156 and 166 and summarized some of the data for single and double mutants at positions 156 and 166 with various substrates.

K166 is a common replacement amino acid in the 156/166 mutants described herein. The replacement of Lys for Gly166 was achieved by using the synthetic DNA cassette at the top right of FIG. 21 which contained the triplet AAA for NNN. This produced fragment 2 with Lys substituting for Gly166.

The 156 substituents were Gln and Ser. The Gln and Ser substitutions at Gly156 are contained within fragment 3 (bottom right FIG. 21).

The multiple mutants were produced by combining fragments 1, 2 and 3 as described in Example 8. The mutants Q156/K166 and S156/K166 were selectively generated by differential phosphorylation as described. Alternatively, the double 156/166 mutants, c.f. Q156/K166 and S156/K166, were prepared by ligation of the 4.6 kb SacI-BamHI fragment from the relevant p156 plasmid containing the 0.6 kb SacI-BamHI fragment from the relevant p166 plasmid.

These mutants, the single mutant K166, and the S156 and Q156 mutants of Example 8 were analyzed for substitute specificity against synthetic polypeptides containing Phe or Glu as the P-1 substrate residue. The results are presented in Table XIII.

                                      TABLE XIII                                   __________________________________________________________________________                Substrate                                                                      P-1               kcat/K.sub.M (mutant)                             Enzymes Compared.sup.(b)                                                                  Residue                                                                             kcat                                                                              K.sub.M                                                                             kcat/K.sub.M                                                                        kcat/K.sub.M (wt)                                 __________________________________________________________________________     Glu-156/Gly-166 (WT)                                                                      Phe  50.00                                                                             1.4 × 10.sup.-4                                                               3.6 × 10.sup.5                                                                (1)                                                          Glu  0.54                                                                              3.4 × 10.sup.-2                                                               1.6 × 10.sup.1                                                                (1)                                               Lys-166    Phe  20.00                                                                             4.0 × 10.sup.-5                                                               5.2 × 10.sup.5                                                                1.4                                                          Glu  0.70                                                                              5.6 × 10.sup.-5                                                               1.2 × 10.sup.4                                                                750                                               Gln-156/Lys-166                                                                           Phe  30.00                                                                             1.9 × 10.sup.-5                                                               1.6 × 10.sup.6                                                                4.4                                                          Glu  1.60                                                                              3.1 × 10.sup.-5                                                               5.0 × 10.sup.4                                                                3100                                              Ser-156/Lys-166                                                                           Phe  30.00                                                                             1.8 × 10.sup.-5                                                               1.6 × 10.sup.6                                                                4.4                                                          Glu  0.60                                                                              3.9 × 10.sup.-5                                                               1.6 × 10.sup.4                                                                1000                                              Ser-156    Phe  34.00                                                                             4.7 × 10.sup.-5                                                               7.3 × 10.sup.5                                                                2.0                                                          Glu  0.40                                                                              1.8 × 10.sup.-3                                                               1.1 × 10.sup.2                                                                6.9                                               Glu-156    Phe  48.00                                                                             4.5 × 10.sup.-5                                                               1.1 × 10.sup.6                                                                3.1                                                          Glu  0.90                                                                              3.3 × 10.sup.-3                                                               2.7 × 10.sup.2                                                                17                                                __________________________________________________________________________

As can be seen in Table XIV, either of these single mutations improve enzyme performance upon substrates with glutamate at the P-1 enzyme binding site. When these single mutations were combined, the resulting multiple enzyme mutants are better than either parent. These single or multiple mutations also alter the relative pH activity profiles of the enzymes as shown in FIGS. 23A and 23B.

To isolate the contribution of electrostatics to substrate specificity from other chemical binding forces, these various single and double mutants were analyzed for their ability to bind and cleave synthetic substrates containing Glu, Gln, Met and Lys as the P-b 1 substrate amino acid. This permitted comparisons between side-chains that were more sterically similar but differed in charge (e.g., Glu versus Gln, Lys versus Met). Similarly, mutant enzymes were assayed against homologous P-1 substrates that were most sterically similar but differed in charge (Table XIV).

                                      TABLE XIV                                    __________________________________________________________________________     Kinetics of Position 156/166 Subtilisins                                       Determined for Different P1 Substrates                                         Enzyme  Net  P-1 Substrate log kcat/K.sub.M (log 1/K.sub.M).sup.(c)            Position.sup.(a)                                                                       Charge.sup.(b)                                                                      Glu   Gln   Met   Lys                                             __________________________________________________________________________     156                                                                               166                                                                         Glu                                                                               Asp  -2   n.d.  3.02 (2.56)                                                                          3.93 (2.74)                                                                          4.23 (3.00)                                     Glu                                                                               Glu  -2   n.d.  3.06 (2.91)                                                                          3.86 (3.28)                                                                          4.48 (3.69)                                     Glu                                                                               Asn  -1   1.62 (2.22)                                                                          3.85 (3.14)                                                                          4.99 (3.85)                                                                          4.15 (2.88)                                     Glu                                                                               Gln  -1   1.20 (2.12)                                                                          4.36 (3.64)                                                                          5.43 (4.36)                                                                          4.10 (3.15)                                     Gln                                                                               Asp  -1   1.30 (1.79)                                                                          3.40 (3.08)                                                                          4.94 (3.87)                                                                          4.41 (3.22)                                     Ser                                                                               Asp  -1   1.23 (2.13)                                                                          3.41 (3.09)                                                                          4.67 (3.68)                                                                          4.24 (3.07)                                     Glu                                                                               Met  -1   1.20 (2.30)                                                                          3.89 (3.19)                                                                          5.64 (4.83)                                                                          4.70 (3.89)                                     Glu                                                                               Ala  -1   n.d.  4.34 (3.55)                                                                          5.65 (4.46)                                                                          4.90 (3.24)                                     Glu                                                                               Gly (wt)                                                                            -1   1.20 (1.47)                                                                          3.85 (3.35)                                                                          5.07 (3.97)                                                                          4.60 (3.13)                                     Gln                                                                               Gly  0    2.42 (2.48)                                                                          4.53 (3.81)                                                                          5.77 (4.61)                                                                          3.76 (2.82)                                     Ser                                                                               Gly  0    2.31 (2.73)                                                                          4.09 (3.68)                                                                          5.61 (4.55)                                                                          3.46 (2.74)                                     Gln                                                                               Asn  0    2.04 (2.72)                                                                          4.51 (3.76)                                                                          5.79 (4.66)                                                                          3.75 (2.74)                                     Ser                                                                               Asn  0    1.91 (2.78)                                                                          4.57 (3.82)                                                                          5.72 (4.64)                                                                          3.68 (2.80)                                     Glu                                                                               Arg  0    2.91 (3.30)                                                                          4.26 (3.50)                                                                          5.32 (4.22)                                                                          3.19 (2.80)                                     Glu                                                                               Lys  0    4.09 (4.25)                                                                          4.70 (3.88)                                                                          6.15 (4.45)                                                                          4.23 (2.93)                                     Gln                                                                               Lys  +1   4.70 (4.50)                                                                          4.64 (3.68)                                                                          5.97 (4.68)                                                                          3.23 (2.75)                                     Ser                                                                               Lys  +1   4.21 (4.40)                                                                          4.84 (3.94)                                                                          6.16 (4.90)                                                                          3.73 (2.84)                                     Maximum difference:                                                                         3.5 (3.0)                                                                            1.8 (1.4)                                                                            2.3 (2.23)                                                                           -1.3 (-1.0)                                     log kcat/K.sub.M (log 1/K.sub.M .sup.(d)                                       __________________________________________________________________________      Footnotes to Table XIV:                                                        .sup.(a) B. subtilis, BG 2036, expressing indicated variant sutilisin wer      fermented and enzymes purified as previously described (Estell, et al.         (1985) J. Biol. Chem. 260, 6518-6521). Wild type subtilisin is indicated       (wt) containing Glu156 and Gly166.                                             .sup.(b) Net charge in the P1 binding site is defined as the sume of           charges from positions 156 and 166 at pH 8.6.                                  .sup.(c) Values for kcat(s.sup.-1) and K.sub.M (M) were measured in 0.1M       Tris pH 8.6 at 25° C. as previously described.sup.3 against P1          substrates having the form succinylL-AlaL-AlaL-ProL-[Xp-nitroanilide,          where X is the indicated P1 amino acid. Values for log 1/K.sub.M are show      inside parentheses. All errors in determination of kcat/K.sub.M and            l/K.sub.M are below 5%.                                                        .sup.(d) Because values for Glu156/Asp166(D166) are too small to determin      accurately, the amximum difference taken for GluP1 substrate is limited t      a charge range of +1 to -1 charge change.                                      n.d. = not determined                                                    

The kcat/K_(M) ratios shown are the second order rate constants for the conversion of substrate to product, and represent the catalytic efficiency of the enzyme. These ratios are presented in logarithmic form to scale the data, and because log kcat/K_(M) is proportional to the lowering of transition-state activation energy (ΔG_(T)). Mutations at position 156 and 166 produce changes in catalytic efficiency toward Glu, Gln, Met and Lys P-1 substrates of 3100, 60, 200 and 20 fold, respectively. Making the P-1 binding-site more positively charged [e.g., compare Gln156/Lys166 (Q156/K166) versus Glu156/Met166 (Glu156/M166)] dramatically increased kcat/K_(M) toward the Glu P-1 substrate (up to 3100 fold), and decreased the catalytic efficiency toward the Lys P-1 substrate (up to 10 fold). In addition, the results show that the catalytic efficiency of wild type enzyme can be greatly improved toward any of the four P-1 substrates by mutagenesis of the P-1 binding site.

The changes in kcat/K_(M) are caused predominantly by changes in 1/K_(M). Because 1/K_(M) is approximately equal to 1/Ks, the enzyme-substrate association constant, the mutations primarily cause a change in substrate binding. These mutations produce smaller effects on kcat that run parallel to the effects on 1/K_(M). The changes in kcat suggest either an alteration in binding in the P-1 binding site in going from the Michaelis-complex E·S) to the transition-state complex (E·S≠) as previously proposed (Robertus, J. D., et al. (1972) Biochemistry 11, 2439-2449; Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), or change in the position of the scissile peptide bond over the catalytic serine in the E·S complex.

Changes in substrate preference that arise from changes in the net charge in the P-1 binding site show trends that are best accounted for by electrostatic effects (FIG. 28). As the P-1 binding cleft becomes more positively charged, the average catalytic efficiency increases much more for the Glu P-1 substrate than for its neutral and isosteric P-1 homolog, Gln (FIG. 28A). Furthermore, at the positive extreme both substrates have nearly identical catalytic efficiencies.

In contrast, as the P-1 site becomes more positively charged the catalytic efficiency toward the Lys P-1 substrate decreases, and diverges sharply from its neutral and isosteric homolog, Met (FIG. 28B). The similar and parallel upward trend seen with increasing positive charge for the Met and Glu P-1 substrates probably results from the fact that all the substrates are succinylated on their amino-terminal end, and thus carry a formal negative charge.

The trends observed in log kcat/K_(M) are dominated by changes in the K_(M) term (FIGS. 28C and 28D). As the pocket becomes more positively charged, the log 1/K_(M) values converge for Glu and Gln P-1 substrates (FIG. 28C), and diverge for Lys and Met P-1 substrates (FIG. 28D). Although less pronounced effects are seen in log kcat, the effects of P-1 charge on log kcat parallel those seen in log 1/K_(M) and become larger as the P-1 pocket becomes more positively charged. This may result from the fact that the transition-state is a tetrahedral anion, and a net positive charge in the enzyme may serve to provide some added stabilization to the transition-state.

The effect of the change in P-1 binding-site charge on substrate preference can be estimated from the differences in slopes between the charged and neutral isosteric P-1 substrates (FIG. 28B). The average change in substrate preference (Δlog kcat/K_(M)) between charged and neutral isosteric substrates increases roughly 10-fold as the complementary charge or the enzyme increases (Table XV). When comparing Glu versus Lys, this difference is 100-fold and the change in substrate preference appears predominantly in the K_(M) term.

                  TABLE XV                                                         ______________________________________                                         Differential Effect on Binding Site                                            Charge on log kcat/K.sub.M or (log l/K.sub.M)                                  for P-1 Substrates that Differ in Charge.sup.(a)                               Change in P-1 Binding                                                                        Δlog kcat/K.sub.M                                                                        (Δlog l/K.sub.M)                           Site Charge.sup.(b)                                                                          GluGln     MetLys   GluLys                                       ______________________________________                                         -2 to -1      n.d.       1.2 (1.2)                                                                               n.d.                                         -1 to 0       0.7 (0.6)  1.3 (0.8)                                                                               2.1 (1.4)                                      0 to +1     1.5 (1.3)  0.5 (0.3)                                                                               2.0 (1.5)                                    Avg. change in                                                                               1.1 (1.0)  1.0 (0.8)                                                                               2.1 (1.5)                                    log kcat/K.sub.M or                                                            (log l/K.sub.M per                                                             unit charge change                                                             ______________________________________                                          .sup.(a) The difference in the slopes of curves were taken between the P1      substrates over the charge interval given for log (k(cat/K.sub.M) (FIG.        3A, B) and (log l/K.sub.M) (FIG. 3D, D). Values represent the differentia      effect a charge change has in distinguishing the substrates that are           compared.                                                                      .sup.(b) Charge in P1 binding site is defined as the sum of charges from       positions 156 and 166.                                                   

The free energy of electrostatic interactions in the structure and energetics of salt-bridge formation depends on the distance between the charges and the microscopic dielectric of the media. To dissect these structural and microenvironmental effects, the energies involved in specific salt-bridges were evaluated. In addition to the possible salt-bridges shown (FIGS. 29A and 29B), reasonable salt-bridges can be built between a Lys P-1 substrate and Asp at position 166, and between a Glu P-1 substrate and a Lys at position 166 (not shown). Although only one of these structures is confirmed by X-ray crystalography (Poulos, T. L., et al. (1976) J. Mol. Biol. 257 1097-1103), all models have favorable torsion angles (Sielecki, A. R., et al. (1979) J. Mol. Biol. 134, 781-804), and do not introduce unfavorable van der Waals contacts.

The change in charged P-1 substrate preference brought about by formation of the model salt-bridges above are shown in Table XVI.

                                      TABLE XVI                                    __________________________________________________________________________     Effect of Salt Bridge Formation Between Enzyme                                 and Substrate on P1 Substrate Preference.sup.(a)                                                               Change                                                                  Substrate.sup.(d)                                                                     in Substrate                                                  Enzyme                                                                              P-1  Preference                                                                            Preference                                     Enzymes Compared.sup.(b)                                                                      Position                                                                            Substrates                                                                          Δlog (kcat/K.sub.M)                                                             ΔΔlog (kcat/K.sub.M)               1       2      Changed                                                                             Compared                                                                            1  2   (1-2)                                          __________________________________________________________________________     Glu156/Asp166                                                                          Gln156/Asp166                                                                         156  LysMet                                                                              +0.30                                                                             -0.53                                                                              0.83                                           Glu156/Asn166                                                                          Gln156/Asn166                                                                         156  LysMet                                                                              -0.84                                                                             -2.04                                                                              1.20                                           Glu156/Gly166                                                                          Gln156/Gly166                                                                         156  LysMet                                                                              -0.47                                                                             -2.10                                                                              1.634                                          Glu156/Lsy-166                                                                         Gln156/Lys166                                                                         156  LysMet                                                                              -1.92                                                                             -2.74                                                                              0.82                                           Ave ΔΔlog (kcat/Km) 1.10 ± 0.3                                  Glu156/Asp166                                                                          Glu156/Asn166                                                                         166  LysMet                                                                              +0.30                                                                             -0.84                                                                              1.14                                           Glu156/Glu166                                                                          Glu156/Glu166                                                                         166  LysMet                                                                              +0.62                                                                             -1.33                                                                              1.95                                           Gln156/Asp166                                                                          Gln156/Asn166                                                                         166  LysMet                                                                              -0.53                                                                             -2.04                                                                              1.51                                           Ser156/Asp166                                                                          Ser156/Asn166                                                                         166  LysMet                                                                              -0.43                                                                             -2.04                                                                              1.61                                           Glu156/Lys166                                                                          Glu156/Met166                                                                         166  GluGln                                                                              -0.63                                                                             -2.69                                                                              2/06                                           Ave ΔΔlog (kcat/K.sub.M)                                                                           1.70 ± 0.3                                  __________________________________________________________________________      Footnotes to Table XVI:                                                        .sup.(a) Molecular modeling shows it is possible to form a salt bridge         between the indicated charged P1 substrate and a complementary charge in       the P1 binding site of the enzyme at the indicated position changed.           .sup.(b) Enzymes compared have sterically similar amino acid substitution      that differ in charge at the indicated position.                               .sup.(c) The P1 substrates compared are structurally similar but differ i      charge. The charged P1 substrate is complementary to the charge change at      the position indicated between enzymes 1 and 2.                                .sup.(d) Data from Table XIV was used to compute the difference in log         (kcat/K.sub.M) between the charged and the noncharged P1 substrate (i.e.,      the substrate preference). The substrate preference is shown separately        for enzyme 1 and 2.                                                            .sup.(e) The difference in substrate preference between enzyme 1 (more         highly charged) and enzyme 2 (more neutral) represents the rate change         acoompanying the electrostatic interaction.                              

The difference between catalytic efficiencies (i.e., Δlog kcat/K_(M)) for the charged and neutral P-1 substrates (e.g., Lys minus Met or Glu minus Gln) give the substrate preference for each enzyme. The change in substrate preference (Δlog kcat/K_(M)) between the charged and more neutral enzyme homologs (e.g., Glu156/Gly166 minus Gln156(Q156)/Gly166) reflects the change in catalytic efficiency that may be attributed solely to electrostatic effects.

These results show that the average change in substrate preference is considerably greater when electrostatic substitutions are produced at position 166 (50-fold in kcat/K_(M)) versus position 156 (12-fold in kcat/K_(M)). From these ΔΔlog kcat/K_(M) values, an average change in transition-state stabilization energy can be calculated of -1.5 and -2.4 kcal/mol for substitutions at positions 156 and 166, respectively. This should represent the stabilization energy contributed from a favorable electrostatic interaction for the binding of free enzyme and substrate to form the transition-state complex.

At least three factors can contribute to the higher transition-state binding energies for electrostatic interactions at position 166. These include: (i) smaller charge separation for salt-bridges at position 166; (ii) more stable side-chain geometries for salt-bridges at position 166; and (iii) microscopic dielectric constants at positions 166.

It is unreasonable to expect all of the energy difference to be due to shorter salt bridges at position 166, because these would have to be 1.6 times shorter than at position 156 for which crystallographic data (Mathews, D. A., et al. (1975) J. Biol. Chem. 250, 7120-7126) indicate are optimally formed. Furthermore, molecular models of salt-bridges appear as structurally reasonable at 156 as at 166.

The binding energies may be more easily explained as differences in the microscopic dielectric constants at position 156 and 166. Assuming a salt-bridge distance of 3A, ˜2.7A), the calculated dielectric constant at position 156 would be 72 (ΔGe=Z₁ Z₂ /rD where Z is the charge on particle 1 and 2, r is the charge separation, and D is the dielectric constant). This corresponds closely with the dielectric constant of 78 for water at this temperature, and qualitatively fits with the fact that position 156 is located on the surface of the enzyme, and is freely exposed to solvent. A calculated dielectric constant for a salt-bridge at position 166 is 45, and fits with the fact that position 166 is more buried and less accessible to solvent. Furthermore, our estimate, based on the hydrophobicity, of the P-1 binding site, indicates that P-1 binding site has an overall dielectric constant close to that of ethanol (D=25).

A large number of mutant comparisons is necessary to show a statistically significant difference between salt-bridges at positive 156 and 166 because there is considerable variation in Δlog kcat/K_(M) for different mutant comparisons at the same position. The change in susbtrate preference from putative salt-bridges at position 156 varies from six to 40-fold in kcat/K_(M), and those at position 166 vary 14 to 120 fold.

In addition to variation produced by factors mentioned above, it is possible that the P-1 side chains are not binding in the same ways between the enzymes compared, even though the comparisons are nearly isosteric in each case. For example, the Lys P-1 substrate side chain may contact Glu156 in Glu156/Asp166 (Glu156/D166) and Asp166 in Gln156/Asp166 (Q156/D166). Thus, one salt-bridge may be substitued for another. It is also possible that complementary charges within the P-1 binding site, e.g., Glu156/Lys166 (Glu156/K166), can form an intramolecular salt-bridge so that the charged side-chains are not free to interact independently with the substrate. Given these caveats it is remarkable that greater variation in substrate preference is not seen by electrostatic substitutions at each position.

EXAMPLE 10 Substitutions at Position 217

Tyr217 has been substituted by all other 19 amino acids. Cassette mutagenesis as described in EPO publication No. 0130756 was used according to the protocol of FIG. 22. The EcoRV restriction site was used for restriction-purification of p.sup.Δ 217.

Since this position is involved in substrate binding, mutations here affect kinetic parameters of the enzyme. An example is the substitution of Leu for Tyr at position 217. For the substrate sAAPFpNa, this mutant has a kcat of 277 5' and a K_(M) of 4.7×10⁻⁴ with a kcat/K_(M) ratio of 6×10⁵. This represents a 5.5-fold increase in kcat with a 3-fold increase in K_(M) over the wild type enzyme.

In addition, replacement of Tyr217 by Lys, Arg, Phe or Leu results in mutant enzymes which are more stable at pH of about 9-11 than the WT enzyme. Conversely, replacement of Tyr217 by Asp, Glu, Gly or Pro results in enzymes which are less stable at pH of about 9-11 than the WT enzyme.

EXAMPLE 11 Multiple Mutants Having Altered Thermal Stability

B. amyloliquifaciens subtilisin does not contain any cysteine residues. Thus, any attempt to produce thermal stability by Cys cross-linkage required the substitution of more than one amino acid in subtilisin with Cys. The following subtilisin residues were multiply substituted with cysteine:

Thr22/Ser87

Ser24/Ser87

Mutagenesis of Ser24 to Cys was carried out with a 5' phosphorylated oligonucleotide primer having the sequence

                       **                                                          5'-pC-TAT-ACT-GGA-TGC-AAT-GTT-AAA-G-3'.                                   

(Asterisks show the location of mismatches and the underlined sequence shows the position of the altered Sau3A site.) The B. amyloliquefaciens subtilisin gene on a 1.5 kb EcoRI-BAMHI fragment from pS4.5 was cloned into M13mp11 and single stranded DNA was isolated. This template (M13mp11SUBT) was double primed with the 5' phosphorylated M13 universal sequencing primer and the mutagenesis primer. Adelman, et al. (1983) DNA 2, 183-193. The heteroduplex was transfected into competent JM101 cells and plaques were probed for the mutant sequence (Zoller, M. J., et al. (1982) Nucleic Acid Res. 10, 6487-6500; Wallace, et al. (1981) Nucleic Acid Res. 9, 3647-3656) using a tetramethylammonium chloride hybridization protocol (Wood, et al. (1985) Proc. Natl. Acad. Sci. USA 82, 1585-1588). The Ser87 to Cys mutation was prepared in a similar fashion using a 5' phosphorylated primer having the sequence

                        *                                                          5'-pGGC-GTT-GCG-CCA-TGC-GCA-TCA-CT-3'.                                    

(The asterisk indicates the position of the mismatch and the underlined sequence shows the position of a new MstI site.) The C24 and C87 mutations were obtained at a frequency of one and two percent, respectively. Mutant sequences were confirmed by dideoxy sequencing in M13.

Mutagenesis of Tyr21/Thr22 to A21/C22 was carried out with a 5' phosphorylated oligonucleotide primer having the sequence

                       *** **    *                                                 5'-pAC-TCT-CAA-GGC-GCT-TGT-GGC-TCA-AAT-GTT-3'.                            

(The asterisks show mismatches to the wild type sequence and the underlined sequence shows the position of an altered Sau3A site.) Manipulations for heteroduplex synthesis were identical to those described for C24. Because direct cloning of the heteroduplex DNA fragment can yield increased frequencies of mutagenesis, the EcoRI-BamHI subtilisin fragment was purified and ligated into pBS42. E. coli MM 294 cells were transformed with the ligation mixture and plasmid DNA was purified from isolated transformants. Plasmid DNA was screened for the loss of the Sau3A site at codon 23 that was eliminated by the mutagenesis primer. Two out of 16 plasmid preparations had lost the wild type Sau3A site. The mutant sequence was confirmed by dideoxy sequencing in M13.

Double mutants, C22/C87 and C24/C87, were constructed by ligating fragments sharing a common ClaI site that separated the single parent cystine codons. Specifically, the 500 bp EcoRI-ClaI fragment containing the 5' portion of the subtilisin gene (including codons 22 and 24) was ligated with the 4.7 kb ClaI-EcoRI fragment that contained the 3' portion of the subtilisin gene (including codon 87) plus pBS42 vector sequence. E. coli MM 294 was transformed with ligation mixtures and plasmid DNA was purified from individual transformants. Double-cysteine plasmid constructions were identified by restriction site markers originating from the parent cysteine mutants (i.e., C22 and C24, Sau3A minus; Cys87, MstI plus). Plasmids from E. coli were transformed into B. subtilis BG2036. The thermal stability of these mutants as compared to wild type subtilisin are presented in FIG. 30 and Tables XVII and XVIII.

                  TABLE XVII                                                       ______________________________________                                         Effect of DTT on the Half-Time of                                              Autolytic Inactivation of Wild-Type                                            and Disulfide Mutants of Subtilisin*                                                     t1/2                                                                         -DDT      +DTT                                                         Enzyme      min           -DTT/+DTT                                            ______________________________________                                         Wild-type 95          85      1.1                                              C22/C87   44          25      1.8                                              C24/C87   92          62      1.5                                              ______________________________________                                          (*)Purified enzymes were either treated or not treated with 25 mM DTT and      dialyzed with or without 10 mM DTT in 2 mM CaCl.sub.2, 50 mM Tris (pH 7.5      for 14 hr. at 4° C. Enzyme concentrations were adjusted to 80 μ      aliquots were quenched on ice and assayed for residual activity. Halftime      for autolytic inactivation were determined from semilog plots of               log.sub.10 (residual activity) versus time. These plots were linear for        over 90% of the inactivation.                                            

                  TABLE XVIII                                                      ______________________________________                                         Effect of Mutations in Subtilisin                                              on the Half-Time of Autolytic                                                  Inactivation at 58° C.*                                                                t1/2                                                                    Enzyme min                                                             ______________________________________                                                 Wild-type                                                                             120                                                                     C22    22                                                                      C24    120                                                                     C87    104                                                                     C22/C87                                                                               43                                                                      C24/C87                                                                               115                                                             ______________________________________                                          (*)Half-times for autolytic inactivation were determined for wildtype and      mutant subtilisins as described in the legend to Table III. Unpurified an      nonreduced enzymes were used directly from B. subtilis culture                 supernatants.                                                            

It has been demonstrated that double-cysteine mutants of subtilisin are efficiently secreted and that disulfide bonds are formed in vivo in B. subtilis (date not shown). The introduction of disulfide bonds in subtilisin extends upon previous work in dihydrofolate reductase and T4 lysozyme (Perry, L. J., et al. (1984) Science 226, 555-557), where single cysteines were introduced near pre-existing cysteines and disulfides were oxidized in vitro. Analyses of physical properties of the subtilisin disulfides, unlike the T4 lysozyme disulfide (Perry, L. J., et al. (1986) Biochemistry, in press), were not complicated by the presence of free cysteines other than those involved in disulfide formation. Because most naturally occuring disulfides occur in secreted proteins, subtilisin is an excellent model system to identify the structural requirements for in vitro formation of stable disulfide bonds in secreted proteins.

Thermal Stability and Autolytic Stability--The data presented here do not address reverisble thermostability of subtilisin directly because of complications arising from autolysis and aggregation. For example, studies monitoring the change in the circular dichroic eliptcity at 220 nm versus temperature of phenylmethanesulfonyl fluorideinhibited subtilisin show typical melt profiles that are coincident with the autolysis curves. However, at the end of thermal melt, SDS-PAGE shows that >90% of the subtilisin is autolyzed. Moreover, Brown and Schleich (Brown, M. F., et al. (1975) Biochemistry 14, 3069-3074) have shown that diisopropylfluorophosphate-inhibited subtilisin irreversibly aggregates in denaturants, which precludes reversible denaturation studies. Thus, until these problems are overcome, subtilisin is not an ideal system for studying the thermodynamics of protein folding.

Although there appears to be a relationship between autolytic stability and conformational stability, the disulfides introduced into subtilisin did not improve the autolytic stability of the mutant enzymes when compared to the wild-type enzyme. However, the disulfide bonds did provide a margin of autolytic stability when compared to their corresponding reduced double-cysteine enzyme. Inspection of a highly refined x-ray structure of wild-type B. amylolicuefaciens subtilisin reveals a hydrogen bond between Thr22 and Ser87. Because cysteine is a poor hydrogen donor or acceptor (Paul, I. C. (1974) in Chemistry of the --SH Group (Patai, S., ed.) pp. 111-149, Wiley Interscience, New York) weakening of 22/87 hydrogen bond may explain why the C22 and C87 single-cysteine mutant proteins are less autolytically stable than either C24 or wild-type (Table XVIII). The fact that C22 is less autolytically stable than C87 may be the result of the Tyr21A mutation (Table XVIII). Indeed, recent construction and analysis of Tyr21/C22 shows the mutant protein has an autolytic stability closer to that of C87. In summary, the C22 and C87 of single-cysteine mutations destabilize the protein toward autolysis, and disulfide bond formation increases the stability to a level less than or equal to that of wild-type enzyme.

These data suggest that the stabilizing effect of an engineered disulfide can be lowered when the parent cysteine mutations disrupt pre-existing stabilizing interactions. Similar conclusions have been drawn from reversible thermal unfolding studies of disulfide cross-linked T4 lysozyme mutants that contain destabilizing mutations. Therefore, a strategy to stabilize a protein by introduction of a disulfide bond should consider avoiding the disruption of stabilizing interactions as well as producing a disulfide with good bond geometry.

EXAMPLE 12 Multiple Mutants Containing Substitutions at Position 222 and Position 166 or 169

Double mutants 166/222 and 169/222 were prepared by ligating together (1) the 2.3 kb AcaII fragment from pS4.5 which contains the 5' portion of the subtilisin gene and vector sequences, (2) the 200 bp AvaII fragment which contains the relevant 166 or 169 mutations from the respective 166 or 169 plasmids, and (3) the 2.2 kb AvaII fragment which contains the relevant 222 mutation 3' and of the subtilisin genes and vector sequence from the respective p222 plasmid.

Although mutations at position 222 improve oxidation stability they also tend to increase the K_(M). An example is shown in Table XIX. In this case the A222 mutation was combined with the K166 mutation to give an enzyme with kcat and K_(M) intermediate between the two parent enzymes.

                  TABLE XIX                                                        ______________________________________                                                          kcat   K.sub.M                                                ______________________________________                                         WT               50     1.4 × 10.sup.-4                                  A222             42     9.9 × 10.sup.-4                                  K166             21     3.7 × 10.sup.-5                                  K166/A222        29     2.0 × 10.sup.-4                                  ______________________________________                                          substrate sAAPFpNa                                                       

EXAMPLE 13 Multiple Mutants Containing Substitutions at Positions 50, 156, 166, 217 and Combinations Thereof

The double mutant S156/A169 was prepared by ligation of two fragments, each containing one of the relevant mutations. The plasmid pS156 was cut with XmaI and treated with S1 nuclease to create a blunt end at codon 167. After removal of the nuclease by phenol/chloroform extraction and ethanol precipitation, the DNA was digested with BamHII and the approximately 4 kb fragment containing the vector plus the 5' portion of the subtilisin gene through codon 167 was purified.

The pA169 plasmid was digested with KpnI and treated with DNA polymerase Klenow fragment plus 50 μM dNTPs to create a blunt end codon at codon 168. The Klenow was removed by phenol/chloroform extraction and ethanol precipitation. The DNA was digested with BamHI and the 590 bp fragment including codon 168 through the carboxy terminus of the subtilisin gene was isolated. The two fragments were then ligated to give S156/A169.

Triple and quadruple mutants were prepared by ligating together (1) the 220 bp PvuII/HaeII fragment containing the relevant 156, 166 and/or 169 mutations from the respective p156, p166 and/or p169 double of single mutant plasmid, (2) the 550 bp HaeII/BamHI fragment containing the relevant 217 mutant from the respective p217 plasmid, and (3) the 3.9 kb PvuII/BamHI fragment containing the F50 mutation and vector sequences.

The multiple mutant F50/S156/A169/L217, as well as B. amyloliquefaciens subtilisin, B. lichenformis subtilisin and the single mutant L217 were analyzed with the above synthetic polypeptides where the P-1 amino acid in the substrate was Lys, His, Ala, Gln, Tyr, Phe, Met and Leu. These results are shown in FIGS. 26 and 27.

These results show that the F50/S156/A169/L217 mutant has substrate specificity similar to that of the B. licheniformis enzyme and differs dramatically from the wild type enzyme. Although only data for the L217 mutant are shown, none of the single mutants (e.g., F50, S156 or A169) showed this effect. Although B. licheniformis differs in 88 residue positions from B. amyloliquefaciens, the combination of only these four mutations accounts for most of the differences in substrate specificity between the two enzymes.

EXAMPLE 14 Subtilisin Mutants Having Altered Alkaline Stability

A random mutagenesis technique was used to generate single and multiple mutations within the B. amyloliquefaciens subtilisin gene. Such mutants were screened for altered alkaline stability. Clones having increased (positive) alkaline stability and decreased (negative) alkaline stability were isolated and sequenced to identify the mutations within the subtilisin gene. Among the positive clones, the mutants V107 and R213 were identified. These single mutants were subsequently combined to produce the mutant V107/R213.

One of the negative clones (V50) from the random mutagenesis experiments resulted in a marked decrease in alkaline stability. Another mutant (P50) was analyzed for alkaline stability to determine the effect of a different substitution at position 50. The F50 mutant was found to have a greater alkaline stability than wild type subtilisin and when combined with the double mutant V107/R213 resulted in a mutant having an alkaline stability which reflected the aggregate of the alkaline stabilities for each of the individual mutants.

The single mutant R204 and double mutant C204/R213 were identified by alkaline screening after random cassette mutagenesis over the region from position 197 to 228. The C204/R213 mutant was thereafter modified to produce mutants containing the individual mutations C204 and R213 to determine the contribution of each of the individual mutations. Cassette mutagenesis using pooled oligonucleotides to substitute all amino acids at position 204, was utilized to determine which substitution at position 204 would maximize the increase in alkaline stability. The mutation from Lys213 to Arg was maintained constant for each of these substitutions at position 204.

A. Construction of pB0180, an E. coli--B. subtilis Shuttle Plasmid

The 2.9 kb EcoRI-BamHI fragment from pBR327 (Covarrubias, L., et al. (1981) Gene 13, 25-35) was ligated to the 3.7 kb EcoRI-BamHI fragment of pBD64 (Gryczan, T., et al. (1980) J. Bacteriol. 141, 246-253) to give the recombinant plasmid pB0153. The unique EcoRI recognition sequence in pBD64 was eliminated by digestion with EcoRI followed by treatment with Klenow and deoxynucleotide triphosphates (Maniatis, T., et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Blunt end ligation and transformation yielded pB0154. The unique AvaI recognition sequence in pB0154 was eliminated in a similar manner to yield pB0171. pB0171 was digested with BamHI and PvuII and treated with Klenow and deoxynucleotide triphosphates to create blunt ends. The 6.4 kb fragment was purified, ligated and transformed into LE392 cells (Enquest, L. W., et al. (1977) J. Mol. Biol. 111, 97-120), to yield pB0172 which retains the unique BamHI site. To facilitate subcloning of subtilisin mutants, a unique and silent KpnI site starting at codon 166 was introduced into the subtilisin gene from pS4.5 (Wells, J. A., et al. (1983) Nucleic Acids Res., 11, 7911-7925) by site-directed mutagenesis. The KpnI+ plasmid was digested with EcoRI and treated with Klenow and deoxynucleotide triphosphates to create a blunt end. The Klenow was inactivated by heating for 20 min at 68° C., and the DNA was digested with BamHI. The 1.5 kb blunt EcoRI-BamHI fragment containing the entire subtilisin was ligated with the 5.8 kb NruI-BamHI from pB0172 to yield pBO180. The ligation of the blunt NruI end to the blunt EcoRI end recreated an EcoRI site. Proceeding clockwise around pB0180 from the EcoRI site at the 5' end of the subtilisin gene is the unique BamHI site at the 3' end of the subtilisin gene, the chloramphenicol and neomycin resistance genes and UB110 gram positive replication origin derived from pBD664, the ampicillin resistance gene and gram negative replication origin derived from pBR327.

B. Construction of Random Mutagenesis Library

The 1.5 kb EcoRI-BamHI fragment containing the B. amyloliquefaciens subtilisin gene (Wells et al., 1983) from pB0180 was cloned into M13mp11 to give M13mp11 SUBT essentially as previously described (Wells, J. A., et al. (1986) J. Biol. Chem., 261,6564-6570). Deoxyuridine containing template DNA was prepared according to Kunkel (Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA, 82 488-492). Uridine containing template DNA (Kunkel, 1985) was purified by CsCl density gradients (Maniatis, T. et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). A primer (AvaI)⁻ having the sequence

                         *                                                                5'GAAAAAAGACCCTAGCGTCGCTTA                                         

ending at codon -11, was used to alter the unique AvaI recognition sequence within the subtilisin gene. (The asterisk denotes the mismatches from the wild-type sequence and underlined is the altered AvaI site.) The 5' phosphorylated AvaI primer (˜320 pmol) and ˜40 pmol (˜120 μg) of uridine containing M13mp11 SUBT template in 1.88 ml of 53 mM NaCl, 7.4 mM MgCl2 and 7.4 mM Tris.HCl (pH 7.5) were annealed by heating to 90° C. for 2 min. and cooling 15 min at 24° C. (FIG. 31). Primer extension at 24° C. was initiated by addition of 100 μL containing 1 mM in all four deoxynucleotide triphosphates, and 20 μl Klenow fragment (5 units/l). The extension reaction was stopped every 15 seconds over ten min by addition of 10 μl 0.25 M EDTA (pH 8) to ⁵⁰,¹ aliquots of the reaction mixture. Samples were pooled, phenol chlorophorm extract and DNA was precipitated twice by addition of 2.5 vol 100% ethanol, and washed twice with 70% ethanol. The pellet was dried, and redissolved in 0.4 ml 1 mM EDTA, 10 mM Tris (pH 8). Misincorporation of α-thiodeoxynucleotides onto the 3' ends of the pool of randomly terminated template was carried out by incubating four 0.2 ml solutions each containing one-fourth of the randomly terminated template mixture (˜20 μg), 0.25 mM of a given α-thiodeoxynucleotide triphosphate, 100 units AMV polymerase, 50 mM KCL, 10 mM MgCl₂, 0.4 mM dithiothreitol, and 50 mM Tris (pH 8.3) (Champoux, J. J. (1984) Genetics, 2, 454-464). After incubation at 37° C. for 90 minutes, misincorporation reactions were sealed by incubation for five minutes at 37° C. with 50 mM all four deoxynucleotide triphosphates (pH 8), and 50 units AMV polymerase. Reactions were stopped by addition of 25 mM EDTA (final), and heated at 68° C. for ten min to inactivate AMV polymerase. After ethanol precipitation and resuspension, synthesis of closed circular heteroduplexes was carried out for two days at 14° C. under the same conditions used for the timed extension reactions above, except the reactions also contained 1000 units T4 DNA ligase, 0.5 mM ATP and 1 mM β-mercaptoethanol. Simultaneous restriction of each heteroduplex pool with KpnI, BamHI, and EcoRI confirmed that the extension reactions were nearly quantitative. Heteroduplex DNA in each reaction mixture was methylated by incubation with 80μM S-adenosylmethionine and 150 units dam methylase for 1 hour at 37° C. Methylation reactions were stopped by heating at 68° C. for 15 min. One-half of each of the four methylated heteroduplex reactions were transformed into 2.5 ml competent E. coli JM101 (Messing, J. (1979) Recombinant DNA Tech. Bull., 2, 43-48). The number of independent transformants from each of the four transformations ranged from 0.4-2.0×10⁵. After growing out phage pools, RF DNA from each of the four transformations was isolated and purified by centrifugation through CsCl density gradients. Approximately 2 μg of RF DNA from each of the four pools was digested with EcoRI, BamHI and AvaI. The 1.5 kb EcoRI-BamHI fragment (i.e., Aval resistant) was purified on low gel temperature agarose and ligated into the 5.5 kb EcoRI-BamHI vector fragment of pB0180. The total number of independent transformants from each α-thiodeoxynucleotide misincorporation plasmid library ranged from 1.2-2.4×10⁴. The pool of plasmids from each of the four transformations was grown out in 200 ml LB media containing 12.5 μg/ml cmp and plasmid DNA was purified by centrifugation through CsCl density gradients.

C. Expression and Screening of Subtilisin Point Mutants

Plasmid DNA from each of the four misincorporation pools was transformed (Anagnostopoulos, C., et al. (1967, J. Bacteriol., 81, 741-746) into BG2036, a strain of B. subtilis deficient in extracellular protease genes (Yang, M. Y. et al. (1984) J. Bacteriol., 160, 15-21). For each transformation, 5 μg of DNA produced approximately 2.5×10⁵ independent BG2036 transformants, and liquid culture aliquots from the four libraries were stored in 10% glycerol at 70° C. Thawed aliquots of frozen cultures were plated on LB/5 μg/ml cmp/1.6% skim milk plates (Wells, J. A., et al. (1983) Nucleic Acids Res., 11, 7911-7925), and fresh colonies were arrayed onto 96-well microtiter plates containing 150 l per well LB media plus 12.5 μg/ml cmp. After 1 h at room temperature, a replica was stamped (using a matched 96 prong stamp) onto a 132 mm BA 85 nitrocellulose filter (Schleicher and Scheull) which was layered on a 140 mm diameter LB/cmp/skim milk plate. Cells were grown about 16 h at 30° C. until halos of proteolysis were roughly 5-7 mm in diameter and filters were transferred directly to a freshly prepared agar plate at 37° C. containing only 1.6% skim milk and 50 mM sodium phosphate pH 11.5. Filters were incubated on plates for 3-6 h at 37° C. to produce halos of about 5 mm for wild-type subtilisin and were discarded. The plates were stained for 10 min at 24° C. with Coomassie blue solution (0.25% Coomassie blue (R-250) 25% ethanol) and destained with 25% ethanol, 10% acetic acid for 20 min. Zones of proteolysis appeared as blue halos on a white background on the underside of the plate and were compared to the original growth plate that was similarly stained and destained as a control. Clones were considered positive that produced proportionately larger zones of proteolysis on the high pH plates relative to the original growth plate. Negative clones gave smaller halos under alkaline conditions. Positive and negative clones were restreaked to colony purify and screened again in triplicate to confirm alkaline pH results.

D. Identification and Analysis of Mutant Subtilisins

Plasmid DNA from 5 ml overnight cultures of more alkaline active B. subtilis clones was prepared according to Birnboim and Doly (1979) except that incubation with 2 mg/ml lysozyme proceeded for 5 min at 37° C. to ensure cell lysis and an additional phenol/CHCl₃ extraction was employed to remove contaminants. The 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was ligated into M13mp11 and template DNA was prepared for DNA sequencing (Messing, J., et al. (1982) Gene, 19 269-276). Three DNA sequencing primers ending at codon 26, +95, and +155 were synthesized to match the subtilisin coding sequence. For preliminary sequence identification a single track of DNA sequence, corresponding to the dNTPaS misincorporation library from which the mutant came, was applied over the entire mature protein coding sequence (i.e., a single dideoxyguanosine sequence track was applied to identify a mutant from the dGTPas library). A complete four track of DNA sequence was performed 200 bp over the site of mutagenesis to confirm and identify the mutant sequence (Sanger, F., et al., (1980) J. Mol. Biol., 143, 161-178). Confirmed positive and negative bacilli clones were cultured in LB media containing 12.5 μg/mL cmp and purified from culture supernatants as previously described (Estell, D. A., et al. (1985) J. Biol. Chem., 260, 6518-6521). Enzymes were greater than 98% pure as analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, U.K. (1970), Nature, 227, 680-685), and protein concentrations were calculated from the absorbance at 280 nm ##EQU2## Maturbara, H., et al. (1965), J. Biol. Chem, 240, 1125-1130).

Enzyme activity was measured with 200 g/mL succinyl-L-AlaL-AlaL-ProL-Phep-nitroanilide (Sigma) in 0.1M Tris pH 8.6 or 0.1 M CAPS pH 10.8 at 25° C. Specific activity (μ moles product/min-mg) was calculated from the change in absorbance at 410 nm from production of p-nitroaniline with time per mg of enzyme (E410=8,480 M-1cm-1; Del Mar, E. G., et al. (1979)Anal. Biochem., 99, 316-320). Alkaline autolytic stability studies were performed on purified enzymes (200 μg/mL) in 0.1 M potassium phosphate (pH 12.0) at 37° C. At various times aliquots were assayed for residual enzyme activity (Wells, J. A., et al. (1986) J. Biol. Chem., 261, 6564-6570).

E. Results

1. Optimization and analysis of mutagenesis frequency

A set of primer-template molecules that were randomly 3'-terminated over the subtilisin gene (FIG. 31) was produced by variable extension from a fixed 5'-primer (The primer mutated a unique AvaI site at codon 11 in the subtilisin gene). This was achieved by stopping polymerase reactions with EDTA after various times of extension. The extent and distribution of duplex formation over the 1 kb subtilisin gene fragment was assessed by multiple restriction digestion (not shown). For example, production of new HinII fragments identified when polymerase extension had proceeded past Ile110, Leu233, and Asp259 in the subtilisin gene.

Misincorporation of each dNTPαs at randomly terminated 3' ends by AMV reverse transcriptase (Zakour, R. A., et al. (1982), Nature, 295, 708-710; Zakour, R. A., et al. (1984), Nucleic Acids Res., 12, 6615-6628) used conditions previously described (Champoux, J. J., (1984), Genetics, 2, 454-464). The efficiency of each misincorporation reaction was estimated to be greater than 80% by the addition of each dNTPαs to the AvaI restriction primer, and analysis by polyacrylamide gel electrophoresis (Champoux, J. J., (1984). Misincorporations were sealed by polymerization with all four dNTP's and closed circular DNA was produced by reaction with DNA ligase.

Several manipulations were employed to maximize the yield of the mutant sequences in the heteroduplex. These included the use of a deoxyuridine containing template (Kunkel, T. A. (1985), Proc. Natl. Acad. Sci. USA, 82 488-492; Pukkila, P. J. et al. (1983), Genetics, 104, 571-582), invitro methylation of the mutagenic strand (Kramer, W. et al. (1982) Nucleic Acids Res., 10 6475-6485), and the use of AvaI restriction-selection against the wild-type template strand which contained a unique AvaI site. The separate contribution of each of these enrichment procedures to the final mutagenesis frequency was not determined, except that prior to AvaI restriction-selection roughly one-third of the segregated clones in each of the four pools still retained a wild-type Aval site within the subtilisin gene. After AvaI restriction-selection greater than 98% of the plasmids lacked the wild-type AvaI site.

The 1.5 kb EcoRI-BamHI subtilisin gene fragment that was resistant to AvaI restriction digestion, from each of the four CsCl purified M13 RF pools was isolated on low melting agarose. The fragment was ligated in situ from the agarose with a similarly cut E. coli--B. subtilis shuttle vector, pB0180, and transformed directly into E coli LE392. Such direct ligation and transformation of DNA isolated from agarose avoided loses and allowed large numbers of recombinants to be obtained (>100,000 per μg equivalent of input M13 pool).

The frequency of mutagenesis for each of the four dNTPas misincorporation reactions was estimated from the frequency that unique restriction sites were eliminated (Table XX). The unique restriction sites chosen for this analysis, ClaI, PvuII, and KpnI, were distributed over the subtilisin gene starting at codons 35, 104, and 166, respectively. As a control, the mutagenesis frequency was determined at the PstI site located in the β lactamase gene which was outside the window of mutagenesis. Because the absolute mutagenesis frequency was close to the percentage of undigested plasmid DNA, two rounds of restriction-selection were necessary to reduce the background of surviving uncut wild-type plasmid DNA below the mutant plasmid (Table XX). The background of surviving plasmid from wild-type DNA probably represents the sum total of spontaneous mutations, uncut wild-type plasmid, plus the efficiency with which linear DNA can transform E. coli. Subtracting the frequency for unmutagenized DNA (background) from the frequency for mutant DNA, and normalizing for the window of mutagenesis sampled by a given restriction analysis (4-6 bp) provides an estimate of the mutagenesis efficiency over the entire coding sequence (₋₋ 1000 bp).

                  TABLE XX                                                         ______________________________________                                         α-thiol                          %                                       dNTP    Restriction                                                                             % resistant clones.sup.c                                                                     % resistant                                                                            mutants                                 misincor-                                                                              Site     1st    2nd        clones over                                                                            per                                 porated.sup.(b)                                                                        Selection                                                                               round  round                                                                               Total Background.sup.d                                                                       1000bp.sup.e                        ______________________________________                                         None    PstI     0.32   0.7  0.002 0       --                                  G       PstI     0.33   1.0  0.003 0.001   0.2                                 T       PstI     0.32   <0.5 <0.002                                                                               0       0                                   C       PstI     0.43   3.0  0.013 0.011   3                                   None    ClaI     0.28   5    0.014 0       --                                  G       ClaI     2.26   85   1.92  1.91    380                                 T       ClaI     0.48   31   0.15  0.14    35                                  C       ClaI     0.55   15   0.08  0.066   17                                  None    PvuII    0.08   29   0.023 0       --                                  G       PvuII    0.41   90   0.37  0.35    88                                  T       PvuII    0.10   67   0.067 0.044   9                                   C       PvuII    0.76   53   0.40  0.38    95                                  None    KpnI     0.41   3    0.012 0       --                                  G       KpnI     0.98   35   0.34  0.33    83                                  T       KpnI     0.36   15   0.054 0.042   8                                   C       KpnI     1.47   26   0.38  0.37    93                                  ______________________________________                                          .sup.(a) Mutagenesis frequency is estimated from the frequency for             obtaining mutations that alter unique restriction sites within the             mutagenized subtilisin gene (i.e., ClaI, PvuII, or KpnI) compared to           mutation frequencies of the PstI site, that is outside the window of           mutagenesis.                                                                   .sup.(b) Plasmid DNA was from wildtype (none) or mutagenized by                DNTPαs misincorporation as described.                                    .sup.(c) Percentage of resistant clones was calculated from the fraction       of clones obtained after three fold or gretaer overdigestion of the            plasmid with the indicated restriction enzyme compared to a nondigested        control. Restrictionresistant plasmid DNA from the first round was             subjected to a second round of restrictionselection. The total respresent      the product of the fractions of resistant clones obtained from both round      of selection and gives percentage of restrictionsite  # mutant clones in       the original starting pool. Frequencies were derived from counting at          least 20 colonies and usually greater than 100.                                .sup.(d) Percent resistant clones was calculated by subtracting the            percentage of restrictionresistant clones obtained for wildtype DNA (i.e.      none) from that obtained for mutant DNA.                                       .sup.(e) This extrapolates from the frequency of mutation over each            restriction site to the entire subtilisin gene (˜1 kb). This has         been normalized to the number of possible bases (4-6 bp) within each           restriction site that can be mutagenized by a given misincorporation           event.                                                                   

From this analysis, the average percentage of subtilisin genes containing mutations that result from dGTPas, dCTPαs, or dTTPαs misincorporation was estimated to be 90, 70, and 20 percent, respectively. These high mutagenesis frequencies were generally quite variable depending upon the dNTPas and misincorporation efficiencies at this site. Misincorporation efficiency has been reported to be both dependent on the kind of mismatch, and the context of primer (Champoux, J. J., (1984); Skinner, J. A., et al. (1986) Nucleic Acids Res., 14, 6945-6964). Biased misincorporation efficiency of dGTPαs and dCTPαs over dTTPαs has been previously observed (Shortle, D., et al. (1985), Genetics, 110, 539-555). Unlike the dGTPαs, dCTPαs, and dTTPαs libraries the efficiency of mutagenesis for the dATPαs misincorporation library could not be accurately assessed because 90% of the restriction-resistant plasmids analyzed simply lacked the subtilisin gene insert. This problem probably arose from self-ligation of the vector when the dATPαs mutagenized subtilisin gene was subcloned from M13 into pB0180. Correcting for the vector background, we estimate the mutagenesis frequency around 20 percent in the dATPαs misincorporation library. In a separate experiment (not shown), the mutagenesis efficiencies for dGTPαs and dTTPαs misincorporation were estimated to be around 50 and 30 percent, respectively, based on the frequency of reversion of an inactivating mutation at codon 169.

The location and identity of each mutation was determined by a single track of DNA sequencing corresponding to the misincorporated αthiodeoxy-nucleotide over the entire gene followed by a complete four track of DNA sequencing focused over the site of mutation. Of 14 mutants identified, the distribution was similar to that reported by Shortle and Lin (1985), except we did not observe nucleotide insertion or deletion mutations. The proportion of AG mutations was highest in the G misincorporation library, and some unexpected point mutations appeared in the dTTPαs and dCTPαs libraries.

2. Screening and Identification of Alkaline Stability Mutants of Subtilisin

It is possible to screen colonies producing subtilisin by halos of casein digestion (Wells, J. A. et al. (1983) Nucleic Acids Res., 11, 7911-7925). However, two problems were posed by screening colonies under high alkaline conditions (>pH 11). First, B. subtilis will not grow at high pH, and we have been unable to transform an alkylophilic strain of bacillus. This problem was overcome by adopting a replica plating strategy in which colonies were grown on filters at neutral pH to produce subtilisin and filters subsequently transferred to casein plates at pH 11.5 to assay subtilisin activity. However, at pH 11.5 the casein micells no longer formed a turbid background and thus prevented a clear observation of proteolysis halos. The problem was overcome by briefly staining the plate with Coomassie blue to amplify proteolysis zones and acidifying the plates to develop casein micell turbidity. By comparison of the halo size produced on the reference growth plate (pH 7) to the high pH plate (pH 11.5), it was possible to identify mutant subtilisins that had increased (positives) or decreased (negatives) stability under alkaline conditions.

Roughly 1000 colonies were screened from each of the four misincorporation libraries. The percentage of colonies showing a differential loss of activity at pH 11.5 versus pH 7 represented 1.4, 1.8, 1.4, and 0.6% of the total colonies screened from the thiol dGTPαs, dATPαs, dTTPαs, and dCTPαs libraries, respectively. Several of these negative clones were sequenced and all were found to contain a single base change as expected from the misincorporation library from which they came. Negative mutants included A36, E170 and V50. Two positive mutants were identified as V107 and R213. The ratio of negatives to positives was roughly 50:1.

3. Stability and Activity of Subtilisin Mutants at Alkaline pH

Subtilisin mutants were purified and their autolytic stabilities were measured by the time course of inactivation at pH 12.0 (FIGS. 32 and 33). Positive mutants identified from the screen (i.e., V107 and R213) were more resistant to alkaline induced autolytic inactivation compared to wild-type; negative mutants (i.e., E170 and V50) were less resistant. We had advantageously produced another mutant at position 50 (F50) by site-directed mutagenesis. This mutant was more stable than wild-type enzyme to alkaline autolytic inactivation (FIG. 33) At the termination of the autolysis study, SDS-PAGE analysis confirmed that each subtilisin variant had autolyzed to an extent consistent with the remaining enzyme activity.

The stabilizing effects of V107, R213, and F50 are cumulative. See Table XXI. The double mutant, V107/R213 (made by subcloning the 920 bp EcoRI-KpnI fragment of pB0180V107 into the 6.6 kb EcoRI-KpnI fragment of pB0180R213), is more stable than either single mutant. The triple mutant, F50/V107/R213 (made by subcloning the 735 bp EcoRI-PvuII fragment of pF50 (Example 2) into the 6.8 kb EcoRI-PvuII fragment of pB0180/V107, is more stable than the double mutant V107/R213 or F50. The inactivation curves show a biphasic character that becomes more pronounced the more stable the mutant analyzed. This may result from some destablizing chemical modificationts) (eg., deamidation) during the autolysis study and/or reduced stabilization caused by complete digestion of larger autolysis peptides. These alkaline autolysis studies have been repeated on separately purified enzyme batches with essentially the same results. Rates of autolysis should depend both on the conformational stability as well as the specific activity of the subtilisin variant (Wells, J. A., et al. (1986), J. Biol. Chem., 261, 6564-6570). It was therefore possible that the decreases in autolytic inactivation rates may result from decreases in specific activity of the more stable mutant under alkaline conditions. In general the opposite appears to be the case. The more stable mutants, if anything, have a relatively higher specific activity than wild-type under alkaline conditions and the less stable mutants have a relatively lower specific activity. These subtle effects on specific activity for V107/R213 and F50/V107/R213 are cumulative at both pH 8.6 and 10.8. The changes in specific activity may reflect slight differences in substrate specificity, however, it is noteworthy that only positions 170 and 107 are within 6 Å of a bound model substrate (Robertus, J. D., et al. (1972), Biochemistry 11, 2438-2449).

                  TABLE XXI                                                        ______________________________________                                         Relationship between relative specific acitivity                               at pH 8.6 or 10.8 and alkaline autolytic stability                                                    Alkaline                                                           Relative specific                                                                          autolysis                                                          activity    half-time                                               Enzyme     pH 8.6      pH 10.8 (min)b                                          ______________________________________                                         Wild-type  100 ± 1  100 ± 3                                                                             86                                              Q170        46 ± 1   28 ± 2                                                                             13                                              V107       126 ± 3   99 ± 5                                                                             102                                             R213        97 ± 1  102 ± 1                                                                             115                                             V107/R213  116 ± 2  106 ± 3                                                                             130                                             V50         66 ± 4   61 ± 1                                                                             58                                              F50        123 ± 3  157 ± 7                                                                             131                                             F50/V107/  126 ± 2  152 ± 3                                                                             168                                             R213                                                                           ______________________________________                                          (a) Relative specific activity was the average from triplicate activity        determinations divided by the wildtype value at the same pH. The average       specific activity of wildtype enzyme at pH 8.6 and 10.8 was 70                 μmoles/minmg and 37 μmoles/minmg, respectively.                          (b)Time to reach 50% activity was taken from FIGS. 32 and 33.            

F. Random Cassette Mutagenesis of Residues 197 through 228

Plasmid pΔ222 (Wells, et al. (1985) Gene 34, 315-323) was digested with PstI and BamHI and the 0.4 kb PstI/BamHI fragment (fragment 1, see FIG. 34) purified from a polyacrylamide gel by electroelution (Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The 1.5 kb EcoRI/BamHI fragment from pS4.5 was cloned into M13mp9. Site directed mutagenesis was performed to create the A197 mutant and simultaneously insert a silent SstI site over codons 195-196. The mutant EcoRI/BamHI fragment was cloned back into pBS42. The A197 plasmid was digested with BamHI and SstI and the 5.3 kb BamHI/SstI fragment (fragment 2) was purified from low melting agarose.

Complimentary oligonucleotides were synthesized to span the region from SstI (codons 195-196) to PstI (codons 228-230). These oligodeoxynucleotides were designed to (1) restore codon 197 to the wild type, (2) re-create a silent KpnI site present in pΔ222 at codons 219-220, (3) create a silent SmaI site over codons 210-211, and (4) eliminate the PstI site over codons 228-230 (see FIG. 35). Oligodeoxynucleotides were synthesized with 2% contaminating nucleotides at each cycle of synthesis, e.g., dATP reagent was spiked with 2% dCTP, 2% dGTP, and 2% dTTP. For 97-mers, this 2% poisoning should give the following percentages of non-mutant, single mutants and double or higher mutants per strand with two or more misincorporations per complimentary strand: 14% non-mutant, 28% single mutant, and 57% with ≧2 mutations, according to the general formula ##EQU3## where μ is the average number of mutations and n is a number class of mutations and f is the fraction of the total having that number of mutations. Complimentary oligodeoxynucleotide pools were phosphorylated and annealed (fragment 3) and then ligated at 2-fold molar excess over fragments 1 and 2 in a three-way ligation. E. coli MM294 was transformed with the ligation reaction, the transformation pool grown up over night and the pooled plasmid DNA was isolated. This pool represented 3.4×10⁴ independent transformants. This plasmid pool was digested with PstI and then used to retransform E. coli. A second plasmid pool was prepared and used to transform B. subtilis (BG2036). Approximately 40% of the BG2036 transformants actively expressed subtilisin as judged by halo-clearing on casein plates. Several of the non-expressing transformants were sequenced and found to have insertions or deletions in the synthetic cassettes. Expressing BG2036 mutants were arrayed in microtiter dishes with 150 μl of LB/12.5 μg/mL chloramphenicol (cmp) per well, incubated at 37° C for 3-4 hours and then stamped in duplicate onto nitrocellulose filters laid on LB 1.5% skim milk/5 μg/mL cmp plates and incubated overnight at 33° C. (until halos were approximately 4-8 mm in diameter). Filters were then lifted to stacks of filter paper saturated with 1×Tide commercial grade detergent, 50 mM Na₂ CO₃, pH 11.5 and incubated at 65° C. for 90 min. Overnight growth plates were Commassie stained and destained to establish basal levels of expression. After this treatment, filters were returned to pH7/skim milk/20 μg/mL tetracycline plates and incubated at 37° C. for 4 hours to overnight.

Mutants identified by the high pH stability screen to be more alkaline stable were purified and analyzed for autolytic stability at high pH or high temperature. The double mutant C204/R213 was more stable than wild type at either high pH or high temperature (Table XXII).

This mutant was dissected into single mutant parents (C204 and R213) by cutting at the unique SmaI restriction site (FIG. 35) and either ligating wild type sequence 3' to the SmaI site to create the single C204 mutant or ligating wild type sequence 5' to the SmaI site to create the single R213 mutant. Of the two single parents, C204 was nearly as alkaline stable as the parent double mutant (C204/R213) and slightly more thermally stable. See Table XXII. The R213 mutant was only slightly more stable than wild type under both conditions (not shown).

Another mutant identified from the screen of the 197 to 228 random cassette mutagenesis was R204. This mutant was more stable than wild type at both high pH and high temperature but less stable than C204.

TABLE XXII Stability of subtilisin variants

Purified enzymes (200 μg/mL) were incubated in 0.1M phosphate, pH 12 at 30° C. for alkaline autolysis, or in 2 mM CaCl₂, 50 mM MOPS, pH 7.0 at 62° C. for thermal autolysis. At various times samples were assayed for residual enzyme activity. Inactivations were roughly pseudo-first order, and t_(1/2) gives the time it took to reach 50% of the starting activity in two separate experiments.

    ______________________________________                                                    t1/2        t1/2                                                               (alkaline   (thermal                                                           autolysis)  autolysis)                                                           Exp.   Exp.       Exp. Exp.                                       Subtilisin variant                                                                          #1     #2         #1   #2                                         ______________________________________                                         wild type    30     25         20   23                                         F50/V107/R213                                                                               49     41         18   23                                         R204         35     32         24   27                                         C204         43     46         38   40                                         C204/R213    50     52         32   36                                         L204/R213    32     30         20   21                                         ______________________________________                                    

G. Random Mutagenesis at Codon 204

Based on the above results, codon 204 was targeted for random mutagenesis. Mutagenic DNA cassettes (for codon at 204) all contained a fixed R213 mutation which was found to slightly augment the stability of the C204 mutant.

Plasmid DNA encoding the subtilisin mutant C204/R213 was digested with SstI and EcoRI and a 1.0 kb EcoRI/SstI fragment was isolated by electro-elution from polyacrylamide gel (fragment 1, see FIG. 35). C204/R213 was also digested with SmaI and EcoRI and the large 4.7 kb fragment, including vector sequences and the 3' portion of coding region, was isolated from low melting agarose (fragment 2, see FIG. 36).

Fragments 1 and 2 were combined in four separate three-way ligations with heterophosphorylated fragments 3 (see FIGS. 36 and 37). This heterophosphorylation of synthetic duplexes should preferentially drive the phosphorylated strand into the plasmid ligation product. Four plasmid pools, corresponding to the four ligations, were restricted with SmaI in order to linearize any single cut C204/R213 present from fragment 2 isolation, thus reducing the background of C204/R213. E. coli was then re-transformed with SmaI-restricted plasmid pools to yield a second set of plasmid pools which are essentially free of C204/R213 and any non-segregated heterduplex material.

These second enriched plasmid pools were then used to transform B. subtilis (BG2036) and the resulting four mutant pools were screened for clones expressing subtilisin resistant to high pH/temperature inactivation. Mutants found positive by such a screen were further characterized and identified by sequencing.

The mutant L204/R213 was found to be slightly more stable than the wild type subtilisin. See Table XXII.

Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention. 

What is claimed is:
 1. A substantially pure subtilisin modified by a substitution of an amino acid at a residue position with a different naturally occurring amino acid, said residue position being selected from the group of equivalent amino acid residues of subtilisin naturally produced by Bacillus amyloliquefaciens consisting of Tyr21, Thr22, Ser24, Asp36, Asn77, Ser87, Lys94, Val95, Leu96, Gly110, Asp197, and Ser204, wherein the subtilisin which is modified is selected from the group consisting of subtilisins derived from procaryotes, yeast and fungi.
 2. A modified subtilisin according to claim 1, wherein said residue position is Tyr21.
 3. A modified subtilisin according to claim 2 wherein said subtilisin has an altered thermal stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Tyr21.
 4. A modified subtilisin according to claim 2 wherein the amino acid substituted at the residue position equivalent to Tyr21 is selected from the group consisting of phenylalanine and leucine.
 5. A modified subtilisin according to claim 1, wherein said residue position is Thr22.
 6. A modified subtilisin according to claim 5 wherein said subtilisin has an altered thermal stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Thr22.
 7. A modified subtilisin according to claim 5 wherein the amino acid substituted at said residue position equivalent to Thr22 is selected from the group consisting of cysteine and lysine.
 8. A modified subtilisin according to claim 1, wherein said residue position is Ser24.
 9. A modified subtilisin according to claim 8 wherein said subtilisin has an altered alkaline stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Ser24.
 10. A modified subtilisin according to claim 8 wherein the amino acid substituted at said residue position equivalent to Ser24 is selected from the group consisting of cysteine and alanine.
 11. A modified subtilisin according to claim 1, wherein said residue position is Asp36.
 12. A modified subtilisin according to claim 11 wherein said subtilisin has an altered alkaline stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Asp36.
 13. A modified subtilisin according to claim 11 wherein said subtilisin has an altered thermal stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Asp36.
 14. A modified subtilisin according to claim 11 wherein the amino acid substituted at said residue position equivalent to Asp36 is selected from the group consisting of glycine and alanine.
 15. A modified subtilisin according to claim 1, wherein said residue position is Asn77.
 16. A modified subtilisin according to claim 15 wherein the amino acid substituted at said residue position equivalent to Asn77 is aspartic acid.
 17. A modified subtilisin according to claim 1, wherein said residue position is Ser87.
 18. A modified subtilisin according to claim 12 wherein said subtilisin has an altered thermal stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Ser87.
 19. A modified subtilisin according to claim 17 wherein the amino acid substituted at said residue position equivalent to Ser87 is selected from the group consisting of cysteine and asparagine.
 20. A modified subtilisin according to claim 18, further comprising a substitution at a second residue position wit a different naturally occurring amino acid, said second residue position being selected from the group of equivalent amino acid residues of subtilisin naturally produced by Bacillus amyloliquifaciens consisting of Thr22 and Ser24, wherein said second residue position is substituted with cysteine.
 21. A modified subtilisin according to claim 1, wherein said residue position is Lys94.
 22. A modified subtilisin according to claim 21 wherein the amino acid substituted at said residue position equivalent to Lys94 is cysteine.
 23. A modified subtilisin according to claim 1, wherein said residue position is Val95.
 24. A modified subtilisin according to claim 23 wherein the amino acid substituted at said residue position equivalent to Val95 is cysteine.
 25. A modified subtilisin according to claim 1, wherein said residue position is Leu96.
 26. A modified subtilisin according to claim 1, wherein said residue position is Gly110.
 27. A modified subtilisin according to claim 26 wherein the amino acid substituted at said residue position equivalent to Gly110 is selected from the group consisting of cysteine and arginine.
 28. A modified subtilisin according to claim 1, wherein said residue position is Asp
 197. 29. A modified subtilisin according to claim 28 wherein said subtilisin has an altered alkaline stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Asp197.
 30. A modified subtilisin according to claim 28 wherein the amino acid substituted at said residue position equivalent to Asp197 is selected from the group consisting of glutamic acid and arginine.
 31. A modified subtilisin according to claim 1, wherein said residue position is Ser204.
 32. A modified subtilisin according to claim 31 wherein said subtilisin has an altered thermal stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at said residue position equivalent to Ser204.
 33. A substantially pure subtilisin modified by a substitution of an amino acid at a residue position with a different naturally occurring amino acid, said residue position being equivalent to Met199 of subtilisin naturally produced by Bacillus amyloliquefaciens, wherein said modified subtilisin has altered thermal stability as compared to the same subtilisin having the amino acid naturally occurring at the residue position equivalent to Met199, wherein the subtilisin which is modified is selected from the group consisting of subtilisins derived from procaryotes, yeast and fungi.
 34. A modified subtilisin according to claim 33 wherein the amino acid substituted at said residue position equivalent to Met199 is isoleucine.
 35. A substantially pure subtilisin modified by a substitution of an amino acid at a residue position with a different naturally occurring amino acid, said residue position being selected from the group of equivalent amino acid residues of subtilisin naturally produced by Bacillus amyloliquefaciens consisting of Ala45, Gly46, Ala48 and Ser49, wherein the subtilisin which is modified is selected from the group consisting of subtilisins derived from procaryotes, yeast and fungi.
 36. A modified subtilisin according to claim 35, wherein said residue position is Ala45.
 37. A modified subtilisin according to claim 35, wherein said residue position is Gly46.
 38. A modified subtilisin according to claim 36 wherein the amino acid substituted at the residue position equivalent to Gly46 is valine.
 39. A modified subtilisin according to claim 35, wherein said residue position is Ala48.
 40. A modified subtilisin according to claim 38 wherein the amino acid substituted at the residue position equivalent to Ala48 is selected from the group consisting of glutamic acid, valine and arginine.
 41. A modified subtilisin according to claim 38, wherein said residue position is Ser49.
 42. A modified subtilisin according to claim 38, wherein the amino acid substituted at the residue position equivalent to Ser49 is selected from the group consisting of cysteine and lysine.
 43. A modified subtilisin according to claim 1 or 33 wherein said subtilisin is derived from a Bacillus subtilisin.
 44. A composition comprising a modified subtilisin according to claim 1 or 33 in combination with a detergent. 